Compositions and methods for targeted delivery to cells

ABSTRACT

Described herein are compositions, kits, and methods for potent delivery to a cell of a subject. The cell can be of a particular cell type, such as a basal cell, a ciliated cell, or a secretory cell. In some cases, the cell can be a lung cell of a particular cell type. Also described herein are pharmaceutical compositions comprising a therapeutic or prophylactic agent assembled to a lipid composition. The lipid composition can comprise an ionizable cationic lipid, a phospholipid, and a selective organ targeting lipid. Further described herein are high-potency dosage forms of a therapeutic or prophylactic agent formulated with a lipid composition.

CROSS-REFERENCE

This application claims the benefits of U.S. Provisional Application No.63/164,523 filed Mar. 22, 2021; U.S. Provisional Application No.63/229,497 filed Aug. 4, 2021; and U.S. Provisional Application No.63/305,426 filed on Feb. 1, 2022, each of which is entirely herebyincorporated by reference herein for all purposes.

BACKGROUND

Therapeutic approaches, such as the CRISPR/Cas (clustered regularlyinterspaced short palindromic repeat / CRISPR-associated protein (Cas))technology, often require precise and potent delivery of therapeuticagent(s) to target organ(s) or cell(s), sometimes in a sequencedependent manner. To date, there remains a clear need to accomplishtherapeutically safe and effective lipid-based carriers for achievingclinical outcomes in the context of genetic diseases and many otherapplications, particularly in the context of respiratory diseases.

SUMMARY

The present application generally concerns safe, efficacious, and potentdelivery of a therapeutic or prophylactic agent such as apolynucleotide, a polypeptide, or a small molecule compound in lipidnanoparticles to target cell(s).

In certain aspects, the present application provides a method for potentdelivery to a (e.g., lung) cell of a subject, comprising: administeringto the subject a (e.g., aerosol) composition comprising a therapeuticagent assembled with a lipid composition which comprises: (i) anionizable cationic lipid; and (ii)a selective organ targeting (SORT)lipid separate from the ionizable cationic lipid, wherein (e.g., anamount of) the SORT lipid effects delivery of the therapeutic agent tothe cell of the subject characterized by a (e.g., about 1.1- or 10-fold)greater therapeutic effect compared to that achieved with a referencelipid composition (e.g., without the amount of the SORT lipid). In someembodiments, the lipid composition further comprises a phospholipid.

In another aspect, the present application provides a method for potentdelivery to (e.g., lung) cells of a subject, comprising: administeringto the subject a (e.g., aerosol) composition comprising a therapeuticagent assembled with a lipid composition which comprises: (i) anionizable cationic lipid; and (ii) a selective organ targeting (SORT)lipid separate from the ionizable cationic lipid, wherein (e.g., anamount of) the SORT lipid effects delivery of the therapeutic agent tocells of the subject characterized by a therapeutic effect in a (e.g.,about 1.1-or 10-fold) greater plurality of (e.g., lung) cells comparedto that achieved with a reference lipid composition (e.g., without theamount of the SORT lipid). In some embodiments, the lipid compositionfurther comprises a phospholipid.

In another aspect, the present application provides a method fortargeted delivery to (e.g., lung) cells of a subject, comprising:administering to the subject a (e.g., aerosol) composition comprising atherapeutic agent assembled with a lipid composition which comprises:(i)an ionizable cationic lipid; and (ii) a selective organ targeting(SORT) lipid separate from the ionizable cationic lipid, wherein (e.g.,an amount of) the SORT lipid effects delivery of the therapeutic agentto a greater proportion of cell types as compared to that achieved witha reference lipid composition. In some embodiments, the lipidcomposition further comprises a phospholipid.

In another aspect, the present application provides a method fortargeted delivery to (e.g., lung) cells of a subject, comprising:administering to the subject a (e.g., aerosol) composition comprising atherapeutic agent assembled with a lipid composition which comprises:(i) an ionizable cationic lipid; and(ii) a selective organ targeting(SORT) lipid separate from the ionizable cationic lipid, wherein (e.g.,an amount of) the SORT lipid effects delivery of the therapeutic agentto cells of the subject characterized by a therapeutic effect in a firstplurality of (e.g., lung) cells of a first cell type and in a (e.g.,about 1.1- or 10-fold) greater second plurality of (e.g., lung) cells ofa second cell type. In some embodiments, the lipid composition furthercomprises a phospholipid.

In another aspect, the present application provides a method fortargeted delivery to (e.g., lung) cells of a subject, comprising:administering to the subject a (e.g., aerosol) composition comprising atherapeutic agent assembled with a lipid composition which comprises:(i) an ionizable cationic lipid; and (ii) a selective organ targeting(SORT) lipid separate from the ionizable cationic lipid, wherein (e.g.,an amount of) the SORT lipid effects a delivery of the therapeutic agentto cells of the subject characterized by a (e.g., about 1.1- or 10-fold)greater therapeutic effect in a first (e.g., lung) cell of a first celltype of the subject compared to that in a second (e.g., lung) cell of asecond cell type of the subject, wherein the first cell type isdifferent from the second cell type. In some embodiments, the lipidcomposition further comprises a phospholipid.

In another aspect, the present application provides a pharmaceuticalcomposition comprising a therapeutic agent assembled with a lipidcomposition, which lipid composition comprises: (i) an ionizablecationic lipid; and (ii) a selective organ targeting (SORT) lipidseparate from the ionizable cationic lipid, wherein the SORT lipid isconfigured to effect a delivery of the therapeutic agent characterizedby one or more of the following: (a) a (e.g., 1.1-or 10-fold) greatertherapeutic effect in a (e.g., lung) cell of the subject compared tothat achieved with a reference lipid composition; (b) a therapeuticeffect in a (e.g., 1.1- or 10-fold) greater plurality of (e.g., lung)cells (e.g., of a cell type) of the subject compared to that achievedwith a reference lipid composition; (c) a therapeutic effect in a firstplurality of (e.g., lung) cells of a first cell type and in a (e.g.,1.1- or 10-fold) greater second plurality of (e.g., lung) cells of asecond cell type; and (d) a (e.g., 1.1- or 10-fold) greater therapeuticeffect in a first (e.g., lung) cell of a first cell type of the subjectcompared to that in a second (e.g., lung) cell of a second cell type ofthe subject. In some embodiments, the lipid composition furthercomprises a phospholipid.

In another aspect, the present disclosure provided a high-potency dosageform of a therapeutic agent formulated with a selective organ targeting(SORT) lipid, the dosage form comprising: the therapeutic agentassembled with a lipid composition that comprises: (i) an ionizablecationic lipid; and (ii) the SORT lipid separate from the ionizablecationic lipid, wherein the SORT lipid is present in the dosage form inan amount sufficient to achieve a therapeutic effect at a dose of thetherapeutic agent (e.g., at least about 1.1- or 10-fold) lower than thatrequired with a reference lipid composition. In some embodiments, thelipid composition further comprises a phospholipid.

In another aspect, the present disclosure provided a high-potency dosageform of a therapeutic agent formulated with a selective organ targeting(SORT) lipid, the dosage form comprising: the therapeutic agentassembled with a lipid composition that comprises: (i) an ionizablecationic lipid; and (ii) the SORT lipid separate from the ionizablecationic lipid, wherein the therapeutic agent (e.g., heterologouspolynucleotide) is present in the dosage form at a dose of no more thanabout 2 milligram per kilogram (mg/kg, or mpk) body weight. In someembodiments, the lipid composition further comprises a phospholipid.

In another aspect, the present application provided a method fordelivery to (e.g., lung) basal cells of a subject, comprising: (e.g.,systemically) administering to the subject a therapeutic agent assembledwith a lipid composition that comprises: (i) an ionizable cationiclipid; and (ii) a selective organ targeting (SORT) lipid separate fromthe ionizable cationic lipid, thereby delivering the therapeutic agentto an organ or tissue (e.g., lung) of the subject to result in atherapeutic effect detectable in at least about 5%, 10%, or 15% basalcells in the organ or tissue of the subject.

Provided herein in some embodiments include a method for delivery bynebulization to lung cell(s) of a subject, the method comprising:administering to said subject a (e.g., pharmaceutical) compositioncomprising a therapeutic agent assembled with a lipid composition, whichlipid composition comprises: (i) an ionizable cationic lipid; and (ii) aselective organ targeting (SORT) lipid separate from said ionizablecationic lipid, thereby delivering said therapeutic agent to said lungcell(s) of a lung of said subject. In some embodiments, the methodprovides a (e.g., therapeutically) effective amount or activity of saidtherapeutic agent in at least about 5%, 10%, 15%, or 20% lung epithelialcells of said subject. In some embodiments, the method provides a (e.g.,therapeutically) effective amount or activity of said therapeutic agentin at least about 2%, 5%, or 10% lung ciliated cells of said subject. Insome embodiments, the method provides a (e.g., therapeutically)effective amount or activity of said therapeutic agent in at least about5%, 10%, 15%, or 20% lung secretory cells of said subject. In someembodiments, the method provides a (e.g., therapeutically) effectiveamount or activity of said therapeutic agent in at least about 5%, 10%,15%, or 20% lung club cells of said subject. In some embodiments, themethod provides a (e.g., therapeutically) effective amount or activityof said therapeutic agent in at least about 5%, 10%, 15%, or 20% lunggoblet cells of said subject. In some embodiments, the method provides a(e.g., therapeutically) effective amount or activity of said therapeuticagent in at least about 5%, 10%, 15%, or 20% lung basal cells of saidsubject. In some embodiments, the lipid composition comprises aphospholipid. In some embodiments, the (e.g., pharmaceutical)composition comprising said therapeutic agent assembled with said lipidcomposition is an aerosol composition.

Additional aspects and advantages of the present application will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent application are shown and described. As will be realized, thepresent application is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the presentinvention will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are utilized, and the accompanying drawings(also “Figure” and “FIG.” herein), of which:

FIG. 1 shows the chemical structures of lipids.

FIG. 2 shows the chemical structures of dendrimer or dendron lipids

FIG. 3 shows a chart of cells type and expression levels of a deliveredmRNA using different compositions of LNP.

FIG. 4 shows images using in vivo imaging of bioluminescence of a mouseafter inhaled aerosol delivery of a Luc mRNA/LNP using multiplecompositions of LNP.

FIG. 5 shows a chart regarding cell toxicity of various LNP compositionsin human bronchial epithelial (hBE) cells.

FIG. 6 shows the stability and general characteristics of various LNPcompositions.

FIG. 7 shows a chart of tissue specific radiance over time in a mouse ofa LNP composition (“Lung-SORT”; 5A2-SC8 DOTAP).

FIG. 8 shows images of tissue specific radiance over time in a mouse ofa LNP composition (“Lung-SORT”; 5A2-SC8 DOTAP).

FIG. 9A shows a workflow for a safety and tolerability study in humansusing a composition described herein.

FIG. 9B illustrates an ex vivo model of ciliated epithelial cells (mousetracheal epithelial cells or MTECs cultured at an air-liquid Interface(ALI) for testing the efficacy of rescue by the DNAI1 mRNA treatmentdescribed herein

FIG. 9C illustrates that ciliary activity in KO mouse cells was rescuedby the DNAI1 mRNA treatment, and the treatment effect remained stablefor weeks after dosing was stopped.

FIG. 10A shows summaries of experiments performed to measure propertiesof lipid compositions described herein.

FIG. 10B shows outcomes of experiments performed to measure propertiesof lipid compositions described herein.

FIG. 10C illustrates positive lung labeling (red) for DNAI1 mRNA (left)and DNAI1 protein (right) in non-human primates using a lipidcomposition (e.g., comprising a SORT lipid) as described herein.

FIG. 10D illustrates that by replacing 100% of U’s in the mRNA withmodified nucleotide m1Ψ minimized cytokine response.

FIG. 10E illustrates data collected from experiments demonstratingfunctional restoration of cilia, tolerability, and selectivity of lipidcompositions described herein.

FIG. 11A shows images of immunofluorescence of human DNAI1 knock-downcells treated with LNPs containing DNAI-HA mRNA. Well-differentiatedhuman DNAI1 knock-down cells were treated with a single dose of aformulation of DNAI1-HA mRNA described herein and immunostained withanti-acetylated tubulin and anti-HA. Integration of newly-expressedDNAI1-HA into axonmeme of cilia peaked between 48 to 72 hours aftertreatment. DNAI1-HA was detected in ciliary axoneme for more than 24days after single administration. Repeated administration resulted inrescue of ciliary activity that remained for weeks after the dosing wasstopped.

FIG. 11B illustrates that newly-made HA-tagged DNAI1 was rapidlyincorporated into the cilia of human bronchial epithelial cells (hBEs).Well-differentiated human DNAI1 knock-down cells were treated (basaladministration) with a single dose of LNP formulated DNAI1-HA (10 µg in2 ml of media). Cells were immunostained with anti-acetylated tubulinand anti-HA 72 hours after dosing. More than 90% of ciliated cells waspositive for DNAI1-HA.

FIG. 12 shows a multiplexed immunofluorescence panel of the respiratoryepithelium of NHPs that may be used to distinguish cell types showingnewly translated DNAI1 protein.

FIG. 13A shows cell tropism signatures of LNP formulations describedherein.

FIG. 13B illustrates aerosol administration of formulated DNAI1 mRNArescued ciliary activity in knock-down primary hBE ALI cultures.Well-differentiated human DNAI1- knock-down cells (hBEs) were treated 2times per week with LNP-formulated DNAi1 (300 µg per Vitrocellnebulization) starting on day 25 post ALI (culture age). Last dose wasadministered on day 50 post ALI. Increased ciliary activity in treatedDNAI1 knock-down cultures was first detected seven days after dosing wasinitiated. Rescued ciliary activity had normal beat frequency (9-17 Hz)and appeared synchronized.

FIGS. 14A-B illustrate cell tropism signatures of a lipid compositioncomprising 20% ionizable cationic lipid (e.g., DODAP) under certainconcentrations and conditions.

FIG. 14C illustrates a cell tropism signature of a lipid compositioncomprising 20 % ionizable cationic lipid (e.g., DODAP) under certainconcentrations and conditions.

FIG. 14D illustrates a cell tropism signature of a lipid compositioncomprising 20% permanently cationic lipid (e.g., 14:0 EPC) under certainconcentrations and conditions.

FIG. 14E illustrates a cell tropism signature of a lipid compositioncomprising 20% permanently cationic lipid (e.g., 14:0 TAP) under certainconcentrations and conditions.

FIG. 15 illustrates cell type related DNAI1 protein expression in targetcells of the respiratory epithelium of NHPs.

FIG. 16A illustrates cell type related DNAI1 protein expression intarget cells of the respiratory epithelium of NHPs.

FIG. 16B shows cell type related DNAI1-HA protein expression in targetcells of the respiratory epithelium of NHPs (left panel, lung; rightpanel bronchi).

FIG. 17 illustrates a study of biodistribution, potency, andtolerability of LNP formulations described herein.

FIG. 18A illustrates the aerosol concentration administered to the NHPs.

FIG. 18B illustrates exemplary measurements of the doses delivered tothe NHPs.

FIG. 18C illustrates characterization of the aerosol composition droplet(MMAD: mass median aerodynamic diameter; GSDL: geometric standarddeviation). The droplet characterization results were within recommendedrange of the Organization for Economic Cooperation and Development(OECD) guidance 433 for inhalation toxicity studies with an MMAD ≤ 4 µmand a GSD between 1.0 and 3.0.

FIGS. 19A-C illustrate measurement of LNP lipid (stemmed from aerosoldroplet) in lung in both low dose and high dose NHP group (FIG. 19A:ionizable lipid in lung; FIG. 19B: DMG-PEG in lung; and FIG. 19C: SORTlipid).

FIG. 20A illustrates DNAI1-HA protein expression in the lung by Westernblotting.

FIG. 20B illustrates DNAI1-HA protein expression in the lung by ELISA.

FIG. 21A illustrates clinical chemistry measurements for AST, ALT, andALP. No significant changes of AST, ALT, or ALP were observed followingtreatment with a lipid composition described herein.

FIG. 21B illustrates the hematology counts of white blood cells andneutrophils. Some increase in neutrophils was observed in thepost-treatment measurements of both vehicle and RTX0052 groups.

FIG. 21C illustrates BAL cell differentials. For cytokine and complementanalysis, cytokines levels were measured in NHP serum and BAL. Analytesmeasured included IFN-α2a, IFN-γ, IL-1β, IL-4, IL-6, IL-10, IL-17A,IP-10, MCP-1, and TNFα. All cytokine levels were in the same range asnormal reported elves.

FIG. 21D illustrates exemplary measurements of cytokine in serum.

FIG. 21E illustrates exemplary measurements of cytokine in BAL.

FIG. 21F illustrates exemplary complement measures of C3a and sC5b-9measurements in plasma and serum respectively.

FIG. 21G illustrates exemplary complement measures of C3a and sC5b-9measurements in BAL.

FIG. 22A illustrates the aerosol concentration administered to the rats.

FIG. 22B illustrates exemplary measurements aerosol homogeneity acrossthree stages.

FIG. 22C illustrates the amount of doses delivered to the rats.

FIG. 22D illustrates characterization of the aerosol composition droplet(MMAD: mass median aerodynamic diameter; GSDL: geometric standarddeviation).

FIGS. 23A-C illustrate measurement of LNP lipid (stemmed from aerosoldroplet) in lung in low dose, mid dose, and high dose rat group (FIG.23A: ionizable lipid in lung; FIG. 23B: DMG-PEG in lung; and FIG. 23C:SORT lipid).

FIG. 24A illustrates DNAI1-HA protein expression in the rat lung byWestern blotting. Six out of ten lung samples I the 1.2 mg/kg, 6 hourgroup were positive for DNAI1-HA.

FIG. 24B illustrates DNAI1-HA protein expression in the rat lung byELISA.

FIG. 25A illustrates clinical chemistry measurements for AST, ALT, andALP in the treated rats.

FIG. 25B illustrates the hematology counts of white blood cells andneutrophils in the treated rats.

FIG. 25C illustrates BAL cell differentials in the treated rats.

FIG. 25D illustrates exemplary measurements of alpha-2-macroglobulin inthe treated rats.

FIG. 26A illustrates the information relating to the four groups of miceto be repeatedly treated with nebulization of LNP/DNAI1-HA mRNA.

FIG. 26B illustrates the protocol for the dosing, imaging, and necropsyof the repeatedly dosed mice.

FIGS. 27A-B illustrate whole body in vivo imaging (IVIS) of therepeatedly dosed mice. Animals, B6 Albino, male, about 7 weeks of age,naïve, were administered 4.0 mg of LNP-formulated DNAI1-HA/Luciferase bynebulization in 2 hours at 66.6 µL/min with Zero grade dry air flow at 2L/min. 4 hour post-dosing, two mice were administered 2 mL of luciferin(30 mg/mL) by nebulization and imaged on IVIS within 1-15 minpost-luciferin administration. Pseudo coloring was applied on the samescale for all images. Lung signal was plotted in graph of FIG. 27A.Whole body signal is plotted in the graph of FIG. 27B.

FIG. 27C illustrates histopathology results of the repeatedly dosedmice.

FIG. 27D illustrates qPCR results showing the relative abundance ofDNAI1-HA mRNA. After the last imaging of the last dose (dose 8), 2 miceper group were perfused.

FIG. 27E illustrates Western blotting showing the protein expression ofDNAI1-HA.

FIG. 28A illustrates delivery of 0.4 mg/kg of LNP-formulated DNAI1 mRNAby inhalation. NHPs were intubated, ventilated, and dosed for fewer than30 minutes.

FIG. 28B illustrates LNP formulation aerosol characteristics. Aerosolparticle size ranges for all three formulations were appropriate fordeposition in the conducting airways.

FIG. 28C illustrates biodistribution of DNAI1-HA mRNA in the targetedcells.

FIG. 28D illustrates DNAI1-HA mRNA ISH results by H-Score. ISH resultsdemonstrated high levels of DNAI1-HA mRNA were delivered to lung cellswith lower levels in the bronchi and trachea.

FIGS. 29A-D illustrate delivery of high levels of DNAI1-HA mRNA to thelung without exposure to liver, spleen, or blood. Digital PCR was usedto measure DNAI1-HA mRNA levels in whole blood, lung, liver, and spleentissue following a single 0.4 mg/kg administration. High levels ofDNAI1-HA mRNA were detected in all three lung regions sampled at 6 hourspost-exposure with RTX0051 and RTX0052. No DNAI1-HA mRNA was detectedabove background in spleen (6 hours, FIG. 29B), liver (6 hours, FIG.29C), or whole blood (30 minutes or 60 minutes, FIG. 29D).

FIG. 30A illustrates multiplex immunofluorescent (IF) images forepithelial cell types.

FIG. 30B illustrates multiplex IF analysis demonstrating expression ofDNAI1-HA protein in target cells in lung.

FIG. 30C illustrates multiplex IF analysis demonstrating expression ofDNAI1-HA protein in target cells in lung with RTX0051.

FIG. 30D illustrates multiplex IF analysis demonstrating expression ofDNAI1-HA protein in target cells in bronchi.

FIG. 30E illustrates multiplex IF analysis demonstrating expression ofDNAI1-HA protein in target cells in bronchi with RTX0051.

FIG. 30F illustrates multiplex IF analysis demonstrating expression ofDNAI1-HA protein in target cells in trachea.

FIGS. 31A-E illustrate BAL cytokine and complement results.

FIGS. 32A-E illustrate plasma cytokine results.

FIG. 33 illustrates transient increase in neutrophils observed in BALand blood at six hours post-exposure.

FIG. 34 illustrates selected clinical chemistry results. Small increaseswere observed for AST, LDH, and creatine kinase in individual animalsafter treatment.

FIG. 35 illustrates summary of tolerability as determined by clinicalobservations and organ weights for the single dose inhalation study.

FIG. 36A illustrates a diagram of the aerosol delivery system. Theamount of aerosolized drug delivered past the endotracheal tube wasestimated using the test setup shown on the left. Pre-weighed glassfiber and MCE filters were attached directly at the exit of theendotracheal tube. Multiple collections were performed before, duringand after treatment of the animals. The glass filters were dried andquantified using both gravimetric analysis. The MCE filters wereanalyzed for amount of mRNA using a RiboGreen assay.

FIG. 36B illustrates the results of aerosol particle size measurements.Particle sizes for test article exposure were measured for deposition inthe conducting airways (branching generations 0-15 in humans).

FIG. 37 illustrates DBAI1-HA mRNA dose present in the NHP. A dashedblack horizontal line represents the targeted presented dose of 0.1mg/kg. Open yellow circles show filter collections before and afterdosing (GF, n=2) using RTX0001/DNAI1-HA mRNA. Open yellow squares showfilter collections before and after dosing (GF, n=2) usingRTX0004/DNAI1-HA mRNA. Similar results obtained for mRNA using MCEfilters and a RiboGreen assay.

FIG. 38A illustrates DNAI1-HA mRNA ISH results for lung tissue. Datafrom assay qualification: 1 of 4 samples per animal analyzed. DNAI1-HAmRNA detected in all animals.

FIG. 38B illustrates that a significant fraction of lung cells containedDNAI1-HA mRNA after treatment with RTX0001 as measured by ISH and thebin scoring.

FIG. 38C illustrates the imaging of the lung tissue used for the ISHanalysis.

FIG. 39A illustrates that the delivery of high levels of DNAI1-HA to thelung did not lead to similar deliver to liver or spleen. Digital PCR wasused to measure DNAI1-HA mRNA levels in whole blood, lung, liver, andspleen tissue following a single 0.1 mg/kg administration. High levelsof DNAI1-HA mRNA were detected in all three lung regions sampled at 6hour post-exposure with RTX0001. In spleen and liver, DNAI1-HA mRNA wasonly measured at or below the LLOQ of the assay.

FIG. 39B illustrates the positive staining of DNAI1-HA tagged protein inNHPs. For RTX0001, DNAI1-HA was detected six hours or 24 hours afteradministration. Regions with higher mRNA levels correlated with regionsshowing highest levels of DNAI1-HA protein. DNAI1-HA mRNA was present inall eight treated animals. No signal detected in vehicle treatedanimals. mRNA levels were highest at six hours and lower at 24 hours.

FIG. 39C illustrates multiplex IF panel for key epithelial cell types.10 NHP FFPE lung tissue blocks (1 from each animal) were used for mIFassay qualification. Two slides from each block were stained induplicates. The cell counts of single marker positive cells, doublepositive cells with DNAI1 expression, and DNAI1 MFI in double positivecells were reported.

FIG. 40A illustrates multiplex IF panel results for NHP lung samples.DNAI1-HA was expressed in cells of the respiratory epithelium.Percentage of DNAI1-HA positive cell was calculated by combining cellcounts from 1 examined lung section per animal. DNAI1-HA expression wasdetected in lung samples from NHPs treated with RTX0001. DNAI1-HAexpression was co-localized with markers for epithelial cells, includingthe club, basal and ciliated cells (club and basal cells are precursorsfor ciliated cells). No staining detected was in lung samples from NHPstreated with RTX0004.

FIG. 40B illustrates multiplex IF analysis of expression of DNAI1-HAprotein in target cell in the lung. Single dose of 0.1 mg/kg ofRTX0001/DNAI1-HA mRNA was administered via inhalation. Lung sectionswere collected from two NHPs at six hours and 24 hours after dosing.Percentage of DNAI1-HA positive cell was calculated by combining cellcounts from all 4 examined lung sections for an individual animal. Totalnumber of cells counted per animal was about 690,000 to 1,100,000. Shownare the individual data points for each treated animal and the mean ±std. dev. for each group (N=2).

DETAILED DESCRIPTION

Before the embodiments of the disclosure are described, it is to beunderstood that such embodiments are provided by way of example only,and that various alternatives to the embodiments of the disclosuredescribed herein may be employed in practicing the invention. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Numerous variations, changes, and substitutions will nowoccur to those skilled in the art without departing from the invention.

In the context of the present application, the following terms have themeanings ascribed to them unless specified otherwise:

As used throughout the specification and claims, the terms “a”, “an” and“the” are generally used in the sense that they mean “at least one”, “atleast a first”, “one or more” or “a plurality” of the referencedcomponents or steps, except in instances wherein an upper limit isthereafter specifically stated. For example, a “cleavage sequence”, asused herein, means “at least a first cleavage sequence” but includes aplurality of cleavage sequences. The operable limits and parameters ofcombinations, as with the amounts of any single agent, will be known tothose of ordinary skill in the art in light of the present application.

The terms “polypeptide”, “peptide”, and “protein” are usedinterchangeably herein to generally refer to polymers of amino acids ofany length. The polymer may be linear or branched, it may comprisemodified amino acids, and it may be interrupted by non-amino acids. Theterms also encompass an amino acid polymer that has been modified, forexample, by disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component.

As used herein in the context of the structure of a polypeptide,“N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxylterminus”) generally refer to the extreme amino and carboxyl ends of thepolypeptide, respectively.

The term “N-terminal end sequence,” as used herein with respect to apolypeptide or polynucleotide sequence of interest, generally means thatno other amino acid or nucleotide residues precede the N-terminal endsequence in the polypeptide or polynucleotide sequence of interest atthe N-terminal end. The term “C-terminal end sequence,” as used hereinwith respect to a polypeptide or polynucleotide sequence of interest,generally means that no other amino acid or nucleotide residues followsthe C-terminal end sequence in the polypeptide or polynucleotidesequence of interest at the C-terminal end.

The terms “non-naturally occurring” and “non-natural” are usedinterchangeably herein. The term “non-naturally occurring” or“non-natural,” as used herein with respect to a therapeutic agent orprophylactic agent, generally means that the agent is not biologicallyderived in mammals (including but not limited to human). The term“non-naturally occurring” or “non-natural,” as applied to sequences andas used herein, means polypeptide or polynucleotide sequences that donot have a counterpart to, are not complementary to, or do not have ahigh degree of homology with a wild-type or naturally-occurring sequencefound in a mammal. For example, a non-naturally occurring polypeptide orfragment may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%or even less amino acid sequence identity as compared to a naturalsequence when suitably aligned.

“Physiological conditions” refers to a set of conditions in a livinghost as well as in vitro conditions, including temperature, saltconcentration, pH, that mimic those conditions of a living subject. Ahost of physiologically relevant conditions for use in in vitro assayshave been established. Generally, a physiological buffer contains aphysiological concentration of salt and is adjusted to a neutral pHranging from about 6.5 to about 7.8, and preferably from about 7.0 toabout 7.5. A variety of physiological buffers are listed in Sambrook etal. (2001). Physiologically relevant temperature ranges from about 25°C. to about 38° C., and preferably from about 35° C. to about 37° C.

As used herein, the terms “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably herein. These terms generallyrefer to an approach for obtaining beneficial or desired resultsincluding but not limited to a therapeutic benefit and/or a prophylacticbenefit. By therapeutic benefit is meant eradication or amelioration ofthe underlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms or improvement in one or more clinical parametersassociated with the underlying disorder such that an improvement isobserved in the subject, notwithstanding that the subject may still beafflicted with the underlying disorder. For prophylactic benefit, thecompositions may be administered to a subject at risk of developing aparticular disease, or to a subject reporting one or more of thephysiological symptoms of a disease, even though a diagnosis of thisdisease may not have been made.

A “therapeutic effect” or “therapeutic benefit,” as used herein,generally refers to a physiologic effect, including but not limited tothe mitigation, amelioration, or prevention of disease or an improvementin one or more clinical parameters associated with the underlyingdisorder in humans or other animals, or to otherwise enhance physical ormental wellbeing of humans or animals, resulting from administration ofa polypeptide of the disclosure other than the ability to induce theproduction of an antibody against an antigenic epitope possessed by thebiologically active protein. For prophylactic benefit, the compositionsmay be administered to a subject at risk of developing a particulardisease, a recurrence of a former disease, condition or symptom of thedisease, or to a subject reporting one or more of the physiologicalsymptoms of a disease, even though a diagnosis of this disease may nothave been made.

The terms “therapeutically effective amount” and “therapeuticallyeffective dose”, as used herein, generally refer to an amount of a drugor a biologically active protein, either alone or as a part of apolypeptide composition, that is capable of having any detectable,beneficial effect on any symptom, aspect, measured parameter orcharacteristics of a disease state or condition when administered in oneor repeated doses to a subject. Such effect need not be absolute to bebeneficial. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein.

The term “equivalent molar dose” generally means that the amounts ofmaterials administered to a subject have an equivalent amount of moles,based on the molecular weight of the material used in the dose.

The term “therapeutically effective and non-toxic dose,” as used herein,generally refers to a tolerable dose of the compositions as definedherein that is high enough to cause depletion of tumor or cancer cells,tumor elimination, tumor shrinkage or stabilization of disease withoutor essentially without major toxic effects in the subject. Suchtherapeutically effective and non-toxic doses may be determined by doseescalation studies described in the art and should be below the doseinducing severe adverse side effects.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation.

When used in the context of a chemical group: “hydrogen” means —H;“hydroxy” means —OH; “oxo” means =O; “carbonyl” means —C(═O)—; “carboxy”means —C(═O)OH (also written as —COOH or —CO₂H); “halo” meansindependently —F, —Cl, —Br or —I; “amino” means —NH₂; “hydroxyamino”means —NHOH; “nitro” means —NO₂; imino means =NH; “cyano” means —CN;“isocyanate” means —N═C═O; “azido” means —N₃; in a monovalent context“phosphate” means —OP(O)(OH)₂ or a deprotonated form thereof; in adivalent context “phosphate” means —OP(O)(OH)O— or a deprotonated formthereof; “mercapto” means —SH; and “thio” means =S; “sulfonyl” means—S(O)₂—; “hydroxysulfonyl” means —S(O)₂OH; “sulfonamide” means—S(O)₂NH₂; and “sulfinyl” means —S(O)—.

In the context of chemical formulas, the symbol “-” means a single bond,“=” means a double bond, and “≡” means triple bond. The symbol “

” represents an optional bond, which if present is either single ordouble. The symbol “

” represents a single bond or a double bond. Thus, for example, theformula

includes

And it is understood that no one such ring atom forms part of more thanone double bond. Furthermore, it is noted that the covalent bond symbol“-”, when connecting one or two stereogenic atoms, does not indicate anypreferred stereochemistry. Instead, it covers all stereoisomers as wellas mixtures thereof. The symbol “

”, when drawn perpendicularlyacross a bond (e.g.,

for methyl) indicates a point of attachment of the group. It is notedthat the point of attachment is typically only identified in this mannerfor larger groups in order to assist the reader in unambiguouslyidentifying a point of attachment. The symbol “

” means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol “

” means a single bond where the group attached to the thick end of thewedge is “into the page”. The symbol “

” means a single bond where the geometry around a double bond (e.g.,either E or Z) is undefined. Both options, as well as combinationsthereof are therefore intended. Any undefined valency on an atom of astructure shown in this application implicitly represents a hydrogenatom bonded to that atom. A bold dot on a carbon atom indicates that thehydrogen attached to that carbon is oriented out of the plane of thepaper.

When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed. When a group “R” is depicted as a“floating group” on a fused ring system, as for example in the formula:

then R may replace any hydrogen attached to any of the ring atoms ofeither of the fused rings unless specified otherwise. Replaceablehydrogens include depicted hydrogens (e.g., the hydrogen attached to thenitrogen in the formula above), implied hydrogens (e.g., a hydrogen ofthe formula above that is not shown but understood to be present),expressly defined hydrogens, and optional hydrogens whose presencedepends on the identity of a ring atom (e.g., a hydrogen attached togroup X, when X equals —CH—), so long as a stable structure is formed.In the example depicted, R may reside on either the 5-membered or the6-membered ring of the fused ring system. In the formula above, thesubscript letter “y” immediately following the group “R” enclosed inparentheses, represents a numeric variable. Unless specified otherwise,this variable can be 0, 1, 2, or any integer greater than 2, onlylimited by the maximum number of replaceable hydrogen atoms of the ringor ring system.

For the chemical groups and compound classes, the number of carbon atomsin the group or class is as indicated as follows: “Cn” defines the exactnumber (n) of carbon atoms in the group/class. “C≤n” defines the maximumnumber (n) of carbon atoms that can be in the group/class, with theminimum number as small as possible for the group/class in question,e.g., it is understood that the minimum number of carbon atoms in thegroup “alkenyl_((C≤8))” or the class “alkene_((C≤8))” is two. Comparewith “alkoxy_((C≤10))”, which designates alkoxy groups having from 1 to10 carbon atoms. “Cn-n′” defines both the minimum (n) and maximum number(n′) of carbon atoms in the group. Thus, “alkyl_((C2-10))” designatesthose alkyl groups having from 2 to 10 carbon atoms. These carbon numberindicators may precede or follow the chemical groups or class itmodifies and it may or may not be enclosed in parenthesis, withoutsignifying any change in meaning. Thus, the terms “C5 olefin”,“C5-olefin”, “olefin_((C5))”, and “olefin_(C5)” are all synonymous.

The term “saturated” when used to modify a compound or chemical groupmeans the compound or chemical group has no carbon-carbon double and nocarbon-carbon triple bonds, except as noted below. When the term is usedto modify an atom, it means that the atom is not part of any double ortriple bond. In the case of substituted versions of saturated groups,one or more carbon oxygen double bond or a carbon nitrogen double bondmay be present. And when such a bond is present, then carbon-carbondouble bonds that may occur as part of keto-enol tautomerism orimine/enamine tautomerism are not precluded. When the term “saturated”is used to modify a solution of a substance, it means that no more ofthat substance can dissolve in that solution.

The term “aliphatic” when used without the “substituted” modifiersignifies that the compound or chemical group so modified is an acyclicor cyclic, but non-aromatic hydrocarbon compound or group. In aliphaticcompounds/groups, the carbon atoms can be joined together in straightchains, branched chains, or non-aromatic rings (alicyclic). Aliphaticcompounds/groups can be saturated, that is joined by singlecarbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or morecarbon-carbon double bonds (alkenes/alkenyl) or with one or morecarbon-carbon triple bonds (alkynes/alkynyl).

The term “aromatic” when used to modify a compound or a chemical groupatom means the compound or chemical group contains a planar unsaturatedring of atoms that is stabilized by an interaction of the bonds formingthe ring.

The term “alkyl” when used without the “substituted” modifier refers toa monovalent saturated aliphatic group with a carbon atom as the pointof attachment, a linear or branched acyclic structure, and no atomsother than carbon and hydrogen. The groups —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr or propyl), —CH(CH₃)₂ (i-Pr, ^(i)Pr or isopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂(isobutyl), —C(CH₃)₃ (tert-butyl, t-butyl, t-Bu or ^(t)Bu), and—CH₂C(CH₃)₃ (neo-pentyl) are non-limiting examples of alkyl groups. Theterm “alkanediyl” when used without the “substituted” modifier refers toa divalent saturated aliphatic group, with one or two saturated carbonatom(s) as the point(s) of attachment, a linear or branched acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups —CH₂— (methylene), —CH₂CH₂—,—CH₂C(CH₃)₂CH₂—, and —CH₂CH₂CH₂— are non-limiting examples of alkanediylgroups. An “alkane” refers to the class of compounds having the formulaH-R, wherein R is alkyl as this term is defined above. When any of theseterms is used with the “substituted” modifier one or more hydrogen atomhas been independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂,—CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The following groups are non-limiting examplesof substituted alkyl groups: —CH₂OH, —CH₂Cl, —CF₃, —CH₂CN, —CH₂C(O)OH,—CH₂C(O)OCH₃, —CH₂C(O)NH₂, —CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OC(O)CH₃, —CH₂NH₂,—CH₂N(CH₃)₂, and —CH₂CH₂Cl. The term “haloalkyl” is a subset ofsubstituted alkyl, in which the hydrogen atom replacement is limited tohalo (i.e. —F, —Cl, —Br, or —I) such that no other atoms aside fromcarbon, hydrogen and halogen are present. The group, —CH₂Cl is anon-limiting example of a haloalkyl. The term “fluoroalkyl” is a subsetof substituted alkyl, in which the hydrogen atom replacement is limitedto fluoro such that no other atoms aside from carbon, hydrogen andfluorine are present. The groups —CH₂F, —CF₃, and —CH₂CF₃ arenon-limiting examples of fluoroalkyl groups.

The term “cycloalkyl” when used without the “substituted” modifierrefers to a monovalent saturated aliphatic group with a carbon atom asthe point of attachment, the carbon atom forming part of one or morenon-aromatic ring structures, no carbon-carbon double or triple bonds,and no atoms other than carbon and hydrogen. Non-limiting examplesinclude: —CH(CH₂)₂ (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl(Cy). The term “cycloalkanediyl” when used without the “substituted”modifier refers to a divalent saturated aliphatic group with two carbonatoms as points of attachment, no carbon-carbon double or triple bonds,and no atoms other than carbon and hydrogen. The group

is a non-limiting example of cycloalkanediyl group. A “cycloalkane”refers to the class of compounds having the formula H-R, wherein R iscycloalkyl as this term is defined above. When any of these terms isused with the “substituted” modifier one or more hydrogen atom has beenindependentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “alkenyl” when used without the “substituted” modifier refersto an monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched, acyclic structure, at leastone nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. Non-limitingexamples include: —CH═CH₂ (vinyl), —CH═CHCH₃, —CH═CHCH₂CH₃, —CH₂CH═CH₂(allyl), —CH₂CH═CHCH₃, and —CH═CHCH═CH₂. The term “alkenediyl” when usedwithout the “substituted” modifier refers to a divalent unsaturatedaliphatic group, with two carbon atoms as points of attachment, a linearor branched, a linear or branched acyclic structure, at least onenonaromatic carbon-carbon double bond, no carbon-carbon triple bonds,and no atoms other than carbon and hydrogen. The groups —CH═CH—,—CH═C(CH₃)CH₂—, —CH═CHCH₂—, and —CH₂CH═CHCH₂— are non-limiting examplesof alkenediyl groups. It is noted that while the alkenediyl group isaliphatic, once connected at both ends, this group is not precluded fromforming part of an aromatic structure. The terms “alkene” and “olefin”are synonymous and refer to the class of compounds having the formulaH-R, wherein R is alkenyl as this term is defined above. Similarly theterms “terminal alkene” and “α-olefin” are synonymous and refer to analkene having just one carbon-carbon double bond, wherein that bond ispart of a vinyl group at an end of the molecule. When any of these termsare used with the “substituted” modifier one or more hydrogen atom hasbeen independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups —CH═CHF, —CH═CHCl and —CH═CHBr arenon-limiting examples of substituted alkenyl groups.

The term “alkynyl” when used without the “substituted” modifier refersto a monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched acyclic structure, at leastone carbon-carbon triple bond, and no atoms other than carbon andhydrogen. As used herein, the term alkynyl does not preclude thepresence of one or more non-aromatic carbon-carbon double bonds. Thegroups —C≡CH, —C≡CCH₃, and —CH₂C≡CCH₃ are non-limiting examples ofalkynyl groups. An “alkyne” refers to the class of compounds having theformula H-R, wherein R is alkynyl. When any of these terms are used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃,—C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “aryl” when used without the “substituted” modifier refers to amonovalent unsaturated aromatic group with an aromatic carbon atom asthe point of attachment, the carbon atom forming part of a one or moresix-membered aromatic ring structure, wherein the ring atoms are allcarbon, and wherein the group consists of no atoms other than carbon andhydrogen. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl or aralkyl groups (carbon number limitation permitting)attached to the first aromatic ring or any additional aromatic ringpresent. Non-limiting examples of aryl groups include phenyl (Ph),methylphenyl, (dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl), naphthyl, anda monovalent group derived from biphenyl. The term “arenediyl” when usedwithout the “substituted” modifier refers to a divalent aromatic groupwith two aromatic carbon atoms as points of attachment, the carbon atomsforming part of one or more six-membered aromatic ring structure(s)wherein the ring atoms are all carbon, and wherein the monovalent groupconsists of no atoms other than carbon and hydrogen. As used herein, theterm does not preclude the presence of one or more alkyl, aryl oraralkyl groups (carbon number limitation permitting) attached to thefirst aromatic ring or any additional aromatic ring present. If morethan one ring is present, the rings may be fused or unfused. Unfusedrings may be connected via one or more of the following: a covalentbond, alkanediyl, or alkenediyl groups (carbon number limitationpermitting). Non-limiting examples of arenediyl groups include:

An “arene” refers to the class of compounds having the formula H-R,wherein R is aryl as that term is defined above. Benzene and toluene arenon-limiting examples of arenes. When any of these terms are used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group -alkanediyl-aryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and2-phenyl-ethyl. When the term aralkyl is used with the “substituted”modifier one or more hydrogen atom from the alkanediyl and/or the arylgroup has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂,—NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃,—NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂. Non-limiting examples of substitutedaralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent aromatic group with an aromatic carbon atom ornitrogen atom as the point of attachment, the carbon atom or nitrogenatom forming part of one or more aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heteroaryl group consists of no atoms other than carbon, hydrogen,aromatic nitrogen, aromatic oxygen and aromatic sulfur. Heteroaryl ringsmay contain 1, 2, 3, or 4 ring atoms selected from are nitrogen, oxygen,and sulfur. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl, aryl, and/or aralkyl groups (carbon number limitationpermitting) attached to the aromatic ring or aromatic ring system.Non-limiting examples of heteroaryl groups include furanyl, imidazolyl,indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl,phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl,quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl,thienyl, and triazolyl. The term “N-heteroaryl” refers to a heteroarylgroup with a nitrogen atom as the point of attachment. The term“heteroarenediyl” when used without the “substituted” modifier refers toan divalent aromatic group, with two aromatic carbon atoms, two aromaticnitrogen atoms, or one aromatic carbon atom and one aromatic nitrogenatom as the two points of attachment, the atoms forming part of one ormore aromatic ring structure(s) wherein at least one of the ring atomsis nitrogen, oxygen or sulfur, and wherein the divalent group consistsof no atoms other than carbon, hydrogen, aromatic nitrogen, aromaticoxygen and aromatic sulfur. If more than one ring is present, the ringsmay be fused or unfused. Unfused rings may be connected via one or moreof the following: a covalent bond, alkanediyl, or alkenediyl groups(carbon number limitation permitting). As used herein, the term does notpreclude the presence of one or more alkyl, aryl, and/or aralkyl groups(carbon number limitation permitting) attached to the aromatic ring oraromatic ring system. Non-limiting examples of heteroarenediyl groupsinclude:

A “heteroarene” refers to the class of compounds having the formula H-R,wherein R is heteroaryl. Pyridine and quinoline are non-limitingexamples of heteroarenes. When these terms are used with the“substituted” modifier one or more hydrogen atom has been independentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH,—OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂,—C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂.

The term “heterocycloalkyl” when used without the “substituted” modifierrefers to a monovalent non-aromatic group with a carbon atom or nitrogenatom as the point of attachment, the carbon atom or nitrogen atomforming part of one or more non-aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heterocycloalkyl group consists of no atoms other than carbon,hydrogen, nitrogen, oxygen and sulfur. Heterocycloalkyl rings maycontain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, orsulfur. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl groups (carbon number limitation permitting) attached tothe ring or ring system. Also, the term does not preclude the presenceof one or more double bonds in the ring or ring system, provided thatthe resulting group remains non-aromatic. Non-limiting examples ofheterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl,piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl,tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl,oxiranyl, and oxetanyl. The term “N-heterocycloalkyl” refers to aheterocycloalkyl group with a nitrogen atom as the point of attachment.N-pyrrolidinyl is an example of such a group. The term“heterocycloalkanediyl” when used without the “substituted” modifierrefers to an divalent cyclic group, with two carbon atoms, two nitrogenatoms, or one carbon atom and one nitrogen atom as the two points ofattachment, the atoms forming part of one or more ring structure(s)wherein at least one of the ring atoms is nitrogen, oxygen or sulfur,and wherein the divalent group consists of no atoms other than carbon,hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present,the rings may be fused or unfused. Unfused rings may be connected viaone or more of the following: a covalent bond, alkanediyl, or alkenediylgroups (carbon number limitation permitting). As used herein, the termdoes not preclude the presence of one or more alkyl groups (carbonnumber limitation permitting) attached to the ring or ring system. Also,the term does not preclude the presence of one or more double bonds inthe ring or ring system, provided that the resulting group remainsnon-aromatic. Non-limiting examples of heterocycloalkanediyl groupsinclude:

When these terms are used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃,—NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂.

The term “acyl” when used without the “substituted” modifier refers tothe group —C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, alkenyl,aryl, aralkyl or heteroaryl, as those terms are defined above. Thegroups, —CHO, —C(O)CH₃(acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃,—C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂, —C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)CH₂C₆H₅,—C(O)(imidazolyl) are non-limiting examples of acyl groups. A “thioacyl”is defined in an analogous manner, except that the oxygen atom of thegroup —C(O)R has been replaced with a sulfur atom, —C(S)R. The term“aldehyde” corresponds to an alkane, as defined above, wherein at leastone of the hydrogen atoms has been replaced with a —CHO group. When anyof these terms are used with the “substituted” modifier one or morehydrogen atom (including a hydrogen atom directly attached to the carbonatom of the carbonyl or thiocarbonyl group, if any) has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups, —C(O)CH₂CF₃, —CO₂H (carboxyl),—CO₂CH₃ (methylcarboxyl), —CO₂CH₂CH₃, —C(O)NH₂ (carbamoyl), and—CON(CH₃)₂, are non-limiting examples of substituted acyl groups.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples include: —OCH₃ (methoxy), —OCH₂CH₃ (ethoxy),—OCH₂CH₂CH₃, —OCH(CH₃)₂ (isopropoxy), —OC(CH₃)₃ (tert-butoxy),—OCH(CH₂)₂, —O—cyclopentyl, and —O—cyclohexyl. The terms “cycloalkoxy”,“alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”,“heterocycloalkoxy”, and “acyloxy”, when used without the “substituted”modifier, refers to groups, defined as -OR, in which R is cycloalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl,respectively. The term “alkoxydiyl” refers to the divalent group—O—alkanediyl—, —O—alkanediyl—O—, or -alkanediyl-O-alkanediyl-. The term“alkylthio” and “acylthio” when used without the “substituted” modifierrefers to the group -SR, in which R is an alkyl and acyl, respectively.The term “alcohol” corresponds to an alkane, as defined above, whereinat least one of the hydrogen atoms has been replaced with a hydroxygroup. The term “ether” corresponds to an alkane, as defined above,wherein at least one of the hydrogen atoms has been replaced with analkoxy group. When any of these terms is used with the “substituted”modifier one or more hydrogen atom has been independently replaced by—OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃,—OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃,—C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples include: —NHCH₃ and —NHCH₂CH₃.

The term “dialkylamino” when used without the “substituted” modifierrefers to the group —NRR′, in which R and R′ can be the same ordifferent alkyl groups, or R and R′ can be taken together to representan alkanediyl. Non-limiting examples of dialkylamino groups include:—N(CH₃)₂ and —N(CH₃)(CH₂CH₃). The terms “cycloalkylamino”,“alkenylamino”, “alkynylamino”, “arylamino”, “aralkylamino”,“heteroarylamino”, “heterocycloalkylamino”, “alkoxyamino”, and“alkylsulfonylamino” when used without the “substituted” modifier,refers to groups, defined as —NHR, in which R is cycloalkyl, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, andalkylsulfonyl, respectively. A non-limiting example of an arylaminogroup is —NHC₆H₅. The term “alkylaminodiyl” refers to the divalent group-NH-alkanediyl-, —NH—alkanediyl—NH—, or -alkanediyl-NH-alkanediyl-. Theterm “amido” (acylamino), when used without the “substituted” modifier,refers to the group -NHR, in which R is acyl, as that term is definedabove. A non-limiting example of an amido group is -NHC(O)CH₃. The term“alkylimino” when used without the “substituted” modifier refers to thedivalent group =NR, in which R is an alkyl, as that term is definedabove. When any of these terms is used with the “substituted” modifierone or more hydrogen atom attached to a carbon atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups —NHC(O)OCH₃ and —NHC(O)NHCH₃ arenon-limiting examples of substituted amido groups.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the present specification and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by the present application. Generallythe term “about,” as used herein when referring to a measurable valuesuch as an amount of weight, time, dose, etc. is meant to encompass inone example variations of ± 20% or ± 10%, in another example ± 5%, inanother example ± 1%, and in yet another example ± 0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed method.

As used in this application, the term “average molecular weight” refersto the relationship between the number of moles of each polymer speciesand the molar mass of that species. In particular, each polymer moleculemay have different levels of polymerization and thus a different molarmass. The average molecular weight can be used to represent themolecular weight of a plurality of polymer molecules. Average molecularweight is typically synonymous with average molar mass. In particular,there are three major types of average molecular weight: number averagemolar mass, weight (mass) average molar mass, and Z-average molar mass.In the context of this application, unless otherwise specified, theaverage molecular weight represents either the number average molar massor weight average molar mass of the formula. In some embodiments, theaverage molecular weight is the number average molar mass. In someembodiments, the average molecular weight may be used to describe a PEGcomponent present in a lipid.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult. “Effective amount,” “Therapeutically effective amount” or“pharmaceutically effective amount” when used in the context of treatinga patient or subject with a compound means that amount of the compoundwhich, when administered to a subject or patient for treating a disease,is sufficient to effect such treatment for the disease.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained. This quantitative measureindicates how much of a particular drug or other substance (inhibitor)is needed to inhibit a given biological, biochemical or chemical process(or component of a process, i.e. an enzyme, cell, cell receptor ormicroorganism) by half.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate (e.g., non-humanprimate). In certain embodiments, the patient or subject is a human.Non-limiting examples of human subjects are adults, juveniles, infantsand fetuses.

The term “assemble” or “assembled,” as used herein, in context ofdelivery of a payload to target cell(s) generally refers to covalent ornon-covalent interaction(s) or association(s), for example, such that atherapeutic or prophylactic agent be complexed with or encapsulated in alipid composition.

As used herein, the term “lipid composition” generally refers to acomposition comprising lipid compound(s), including but not limited to,a lipoplex, a liposome, a lipid particle. Examples of lipid compositionsinclude suspensions, emulsions, and vesicular compositions.

For example, as used herein, “RTX0001” refers to an example lipidcomposition tested herein. RTX0001 is a 5-component lipid nanoparticlecomposition comprising about 19.05% 4A3-SC7 (ionizable cationic lipid),about 20% DODAP (SORT lipid), about 19.05% DOPE, about 38.9%cholesterol, and about 3.81% DMG-PEG (PEG conjugated lipid), whereineach lipid component is defined as mol% of the total lipid composition.

As another example, as used herein, “RTX0004” refers to an example lipidcomposition tested herein. RTX0004 is a 4-component lipid nanoparticlecomposition comprising about 23.81% 5A2-SC8 (ionizable cationic lipid),about 23.81% DOPE, about 47.62% cholesterol, and about 4.76% DMG-PEG(PEG conjugated lipid), wherein each lipid component is defined as mol%of the total lipid composition.

As another example, as used herein, “RTX0051” refers to an example lipidcomposition tested herein. RTX0051 is a 5-component lipid nanoparticlecomposition comprising about 19.05% 4A3-SC7 (ionizable cationic lipid),about 20% 14:0 EPC (SORT lipid), about 19.05% DOPE, about 38.9%cholesterol, and about 3.81% DMG-PEG (PEG conjugated lipid), whereineach lipid component is defined as mol% of the total lipid composition.

As yet another example, as used herein, “RTX0052” refers to an examplelipid composition tested herein. RTX0052 is a 5-component lipidnanoparticle composition comprising about 19.05% 4A3-SC7 (ionizablecationic lipid), about 20% 14:0 TAP (SORT lipid), about 19.05% DOPE,about 38.9% cholesterol, and about 3.81% DMG-PEG (PEG conjugated lipid),wherein each lipid component is defined as mol% of the total lipidcomposition.

As used herein, the term “detectable” refers to an occurrence of, or achange in, a signal that is directly or indirectly detectable either byobservation or by instrumentation. Typically, a detectable response isan occurrence of a signal wherein the fluorophore is inherentlyfluorescent and does not produce a change in signal upon binding to ametal ion or biological compound. Alternatively, the detectable responseis an optical response resulting in a change in the wavelengthdistribution patterns or intensity of absorbance or fluorescence or achange in light scatter, fluorescence lifetime, fluorescencepolarization, or a combination of the above parameters. Other detectableresponses include, for example, chemiluminescence, phosphorescence,radiation from radioisotopes, magnetic attraction, and electron density

The term “potent” or “potency,” as used herein in connection withdelivery of therapeutic agent(s), generally refers to a greater abilityof a delivery system (e.g., a lipid composition) to achieve or bringabout a desired amount, activity, or effect of a therapeutic agent orprophylactic agent (such as a desired level of translation,transcription, production, expression, or activity of a protein or gene)in cells (e.g., targeted cells) to any measurable extent, e.g., relativeto a reference delivery system. For example, a lipid composition with ahigher potency may achieve a desired therapeutic effect in a greaterpopulation of relevant cells, within a shorter response time, or thatlast a longer period of time.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent application which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this disclosure is not critical, so longas the salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag HelveticaChimica Acta, 2002).

The term “pharmaceutically acceptable carrier,” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a chemical agent.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

A “repeat unit” is the simplest structural entity of certain materials,for example, frameworks and/or polymers, whether organic, inorganic ormetal-organic. In the case of a polymer chain, repeat units are linkedtogether successively along the chain, like the beads of a necklace. Forexample, in polyethylene, —[—CH₂CH₂—]_(n)—, the repeat unit is —CH₂CH₂—.The subscript “n” denotes the degree of polymerization, that is, thenumber of repeat units linked together. When the value for “n” is leftundefined or where “n” is absent, it simply designates repetition of theformula within the brackets as well as the polymeric nature of thematerial. The concept of a repeat unit applies equally to where theconnectivity between the repeat units extends three dimensionally, suchas in metal organic frameworks, modified polymers, thermosettingpolymers, etc. Within the context of the dendrimer or dendron, therepeating unit may also be described as the branching unit, interiorlayers, or generations. Similarly, the terminating group may also bedescribed as the surface group.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers. Chiral molecules contain achiral center, also referred to as a stereocenter or stereogenic center,which is any point, though not necessarily an atom, in a moleculebearing groups such that an interchanging of any two groups leads to astereoisomer. In organic compounds, the chiral center is typically acarbon, phosphorus or sulfur atom, though it is also possible for otheratoms to be stereocenters in organic and inorganic compounds. A moleculecan have multiple stereocenters, giving it many stereoisomers. Incompounds whose stereoisomerism is due to tetrahedral stereogeniccenters (e.g., tetrahedral carbon), the total number of hypotheticallypossible stereoisomers will not exceed 2^(n), where n is the number oftetrahedral stereocenters. Molecules with symmetry frequently have fewerthan the maximum possible number of stereoisomers. A 50:50 mixture ofenantiomers is referred to as a racemic mixture. Alternatively, amixture of enantiomers can be enantiomerically enriched so that oneenantiomer is present in an amount greater than 50%. Typically,enantiomers and/or diastereomers can be resolved or separated usingtechniques known in the art. It is contemplated that that for anystereocenter or axis of chirality for which stereochemistry has not beendefined, that stereocenter or axis of chirality can be present in its Rform, S form, or as a mixture of the R and S forms, including racemicand non-racemic mixtures. As used herein, the phrase “substantially freefrom other stereoisomers” means that the composition contains ≤ 15%,more preferably ≤ 10%, even more preferably ≤ 5%, or most preferably ≤1% of another stereoisomer(s).

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

The above definitions supersede any conflicting definition in anyreference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the disclosure in terms such thatone of ordinary skill can appreciate the scope and practice the presentapplication.

Compositions Lipid Compositions

In one aspect, provided herein is a lipid composition comprising: (i) anionizable cationic lipid; and (iii) a selective organ targeting (SORT)lipid separate from the ionizable cationic lipid. The lipid compositionmay further comprise a phospholipid.

Ionizable Cationic Lipids

In some embodiments of the lipid composition of the present application,the lipid composition comprises an ionizable cationic lipid. In someembodiments, the cationic ionizable lipids contain one or more groupswhich is protonated at physiological pH but may deprotonated and has nocharge at a pH above 8, 9, 10, 11, or 12. The ionizable cationic groupmay contain one or more protonatable amines which are able to form acationic group at physiological pH. The cationic ionizable lipidcompound may also further comprise one or more lipid components such astwo or more fatty acids with C₆-C₂₄ alkyl or alkenyl carbon groups.These lipid groups may be attached through an ester linkage or may befurther added through a Michael addition to a sulfur atom. In someembodiments, these compounds may be a dendrimer, a dendron, a polymer,or a combination thereof.

In some embodiments of the lipid composition of the present application,the ionizable cationic lipids refer to lipid and lipid-like moleculeswith nitrogen atoms that can acquire charge (pKa). These lipids may beknown in the literature as cationic lipids. These molecules with aminogroups typically have between 2 and 6 hydrophobic chains, often alkyl oralkenyl such as C₆-C₂₄ alkyl or alkenyl groups, but may have at least 1or more that 6 tails. In some embodiments, these cationic ionizablelipids are dendrimers, which are a polymer exhibiting regular dendriticbranching, formed by the sequential or generational addition of branchedlayers to or from a core and are characterized by a core, at least oneinterior branched layer, and a surface branched layer. (See Petar R.Dvornic and Donald A. Tomalia in Chem. in Britain, 641-645, August1994.) In other embodiments, the term “dendrimer” as used herein isintended to include, but is not limited to, a molecular architecturewith an interior core, interior layers (or “generations”) of repeatingunits regularly attached to this initiator core, and an exterior surfaceof terminal groups attached to the outermost generation. A “dendron” isa species of dendrimer having branches emanating from a focal pointwhich is or can be joined to a core, either directly or through alinking moiety to form a larger dendrimer. In some embodiments, thedendrimer structures have radiating repeating groups from a central corewhich doubles with each repeating unit for each branch. In someembodiments, the dendrimers described herein may be described as a smallmolecule, medium-sized molecules, lipids, or lipid-like material. Theseterms may be used to described compounds described herein which have adendron like appearance (e.g. molecules which radiate from a singlefocal point).

While dendrimers are polymers, dendrimers may be preferable totraditional polymers because they have a controllable structure, asingle molecular weight, numerous and controllable surfacefunctionalities, and traditionally adopt a globular conformation afterreaching a specific generation. Dendrimers can be prepared bysequentially reactions of each repeating unit to produce monodisperse,tree-like and/or generational structure polymeric structures. Individualdendrimers consist of a central core molecule, with a dendritic wedgeattached to one or more functional sites on that central core. Thedendrimeric surface layer can have a variety of functional groupsdisposed thereon including anionic, cationic, hydrophilic, or lipophilicgroups, according to the assembly monomers used during the preparation.

Modifying the functional groups and/or the chemical properties of thecore, repeating units, and the surface or terminating groups, theirphysical properties can be modulated. Some properties which can bevaried include, but are not limited to, solubility, toxicity,immunogenicity and bioattachment capability. Dendrimers are oftendescribed by their generation or number of repeating units in thebranches. A dendrimer consisting of only the core molecule is referredto as Generation 0, while each consecutive repeating unit along allbranches is Generation 1, Generation 2, and so on until the terminatingor surface group. In some embodiments, half generations are possibleresulting from only the first condensation reaction with the amine andnot the second condensation reaction with the thiol.

Preparation of dendrimers requires a level of synthetic control achievedthrough series of stepwise reactions comprising building the dendrimerby each consecutive group. Dendrimer synthesis can be of the convergentor divergent type. During divergent dendrimer synthesis, the molecule isassembled from the core to the periphery in a stepwise process involvingattaching one generation to the previous and then changing functionalgroups for the next stage of reaction. Functional group transformationis necessary to prevent uncontrolled polymerization. Such polymerizationwould lead to a highly branched molecule that is not monodisperse and isotherwise known as a hyperbranched polymer. Due to steric effects,continuing to react dendrimer repeat units leads to a sphere shaped orglobular molecule, until steric overcrowding prevents complete reactionat a specific generation and destroys the molecule’s monodispersity.Thus, in some embodiments, the dendrimers of G1-G10 generation arespecifically contemplated. In some embodiments, the dendrimers comprise1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derivabletherein. In some embodiments, the dendrimers used herein are G0, G1, G2,or G3. However, the number of possible generations (such as 11, 12, 13,14, 15, 20, or 25) may be increased by reducing the spacing units in thebranching polymer.

Additionally, dendrimers have two major chemical environments: theenvironment created by the specific surface groups on the terminationgeneration and the interior of the dendritic structure which due to thehigher order structure can be shielded from the bulk media and thesurface groups. Because of these different chemical environments,dendrimers have found numerous different potential uses including intherapeutic applications.

In some embodiments of the lipid composition of the present application,the dendrimers or dendrons are assembled using the differentialreactivity of the acrylate and methacrylate groups with amines andthiols. The dendrimers or dendrons may include secondary or tertiaryamines and thioethers formed by the reaction of an acrylate group with aprimary or secondary amine and a methacrylate with a mercapto group.Additionally, the repeating units of the dendrimers or dendrons maycontain groups which are degradable under physiological conditions. Insome embodiments, these repeating units may contain one or more germinaldiethers, esters, amides, or disulfides groups. In some embodiments, thecore molecule is a monoamine which allows dendritic polymerization inonly one direction. In other embodiments, the core molecule is apolyamine with multiple different dendritic branches which each maycomprise one or more repeating units. The dendrimer or dendron may beformed by removing one or more hydrogen atoms from this core. In someembodiments, these hydrogen atoms are on a heteroatom such as a nitrogenatom. In some embodiments, the terminating group is a lipophilic groupssuch as a long chain alkyl or alkenyl group. In other embodiments, theterminating group is a long chain haloalkyl or haloalkenyl group. Inother embodiments, the terminating group is an aliphatic or aromaticgroup containing an ionizable group such as an amine (—NH₂) or acarboxylic acid (—CO₂H). In still other embodiments, the terminatinggroup is an aliphatic or aromatic group containing one or more hydrogenbond donors such as a hydroxide group, an amide group, or an ester.

The cationic ionizable lipids of the present application may contain oneor more asymmetrically-substituted carbon or nitrogen atoms, and may beisolated in optically active or racemic form. Thus, all chiral,diastereomeric, racemic form, epimeric form, and all geometric isomericforms of a chemical formula are intended, unless the specificstereochemistry or isomeric form is specifically indicated. Cationicionizable lipids may occur as racemates and racemic mixtures, singleenantiomers, diastereomeric mixtures and individual diastereomers. Insome embodiments, a single diastereomer is obtained. The chiral centersof the cationic ionizable lipids of the present application can have theS or the R configuration. Furthermore, it is contemplated that one ormore of the cationic ionizable lipids may be present as constitutionalisomers. In some embodiments, the compounds have the same formula butdifferent connectivity to the nitrogen atoms of the core. Withoutwishing to be bound by any theory, it is believed that such cationicionizable lipids exist because the starting monomers react first withthe primary amines and then statistically with any secondary aminespresent. Thus, the constitutional isomers may present the fully reactedprimary amines and then a mixture of reacted secondary amines.

Chemical formulas used to represent cationic ionizable lipids of thepresent application will typically only show one of possibly severaldifferent tautomers. For example, many types of ketone groups are knownto exist in equilibrium with corresponding enol groups. Similarly, manytypes of imine groups exist in equilibrium with enamine groups.Regardless of which tautomer is depicted for a given formula, andregardless of which one is most prevalent, all tautomers of a givenchemical formula are intended.

The cationic ionizable lipids of the present application may also havethe advantage that they may be more efficacious than, be less toxicthan, be longer acting than, be more potent than, produce fewer sideeffects than, be more easily absorbed than, and/or have a betterpharmacokinetic profile (e.g., higher oral bioavailability and/or lowerclearance) than, and/or have other useful pharmacological, physical, orchemical properties over, compounds known in the prior art, whether foruse in the indications stated herein or otherwise.

In addition, atoms making up the cationic ionizable lipids of thepresent application are intended to include all isotopic forms of suchatoms. Isotopes, as used herein, include those atoms having the sameatomic number but different mass numbers. By way of general example andwithout limitation, isotopes of hydrogen include tritium and deuterium,and isotopes of carbon include ¹³C and ¹⁴C.

It should be recognized that the particular anion or cation forming apart of any salt form of a cationic ionizable lipids provided herein isnot critical, so long as the salt, as a whole, is pharmacologicallyacceptable. Additional examples of pharmaceutically acceptable salts andtheir methods of preparation and use are presented in Handbook ofPharmaceutical Salts: Properties, and Use (2002), which is incorporatedherein by reference.

In some embodiments of the lipid composition of the present application,the ionizable cationic lipid is a dendrimer or dendron. In someembodiments, the ionizable cationic lipid comprises an ammonium groupwhich is positively charged at physiological pH and contains at leasttwo hydrophobic groups. In some embodiments, the ammonium group ispositively charged at a pH from about 6 to about 8. In some embodiments,the ionizable cationic lipid is a dendrimer or dendron. In someembodiments, the ionizable cationic lipid comprises at least two C₆-C₂₄alkyl or alkenyl groups.

Dendrimers or Dendrons of Formula (I)

In some embodiments of the lipid composition, the ionizable cationiclipid comprises at least two C₈-C₂₄ alkyl groups. In some embodiments,the ionizable cationic lipid is a dendrimer or dendron further definedby the formula: Core-Repeating Unit-Terminating Group (D-I) wherein thecore is linked to the repeating unit by removing one or more hydrogenatoms from the core and replacing the atom with the repeating unit andwherein:

-   the core has the formula:

-   

-   wherein:    -   X₁ is amino or alkylamino_((C≤12)), dialkylamino_((C≤12)),        heterocycloalkyl_((C≤12)), heteroaryl_((C≤12)), or a substituted        version thereof;    -   R₁ is amino, hydroxy, or mercapto, or alkylamino_((C≤12)),        dialkylamino_((C≤12)), or a substituted version of either of        these groups; and    -   a is 1, 2, 3, 4, 5, or 6; or

-   the core has the formula:

-   

-   wherein:    -   X₂ is N(R₅)_(y);    -   R₅ is hydrogen, alkyl_((C≤18)), or substituted alkyl_((C≤18));        and y is 0, 1, or 2, provided that the sum of y and z is 3;    -   R₂ is amino, hydroxy, or mercapto, or alkylamino_((C≤12)),        dialkylamino_((C≤12)), or a substituted version of either of        these groups;    -   b is 1, 2, 3, 4, 5, or 6; and    -   z is 1, 2, 3; provided that the sum of z and y is 3; or

-   the core has the formula:

-   

-   wherein:    -   X₃ is —NR₆—, wherein R₆ is hydrogen, alkyl_((C≤8)), or        substituted alkyl_((C≤8)), —O—, or alkylaminodiyl_((C≤8)),        alkoxydiyl_((C≤8)), arenediyl_((C≤8)), heteroarenediyl_((C≤8)),        heterocycloalkanediyl_((C≤8)), or a substituted version of any        of these groups;

    -   R₃ and R₄ are each independently amino, hydroxy, or mercapto, or        alkylamino_((C≤12)), dialkylamino_((C≤12)), or a substituted        version of either of these groups; or a group of the formula:        —N(R_(f))_(f)(CH₂CH₂N(R_(c)))_(e)R_(d),

    -   

    -   

    -   

    -   wherein:        -   e and f are each independently 1, 2, or 3; provided that the            sum of e and f is 3;        -   R_(c), R_(d), and R_(f) are each independently hydrogen,            alkyl_((C≤6)), or substituted alkyl_((C≤6));

    -   c and d are each independently 1, 2, 3, 4, 5, or 6; or the core        is alkylamine_((C≤18)), dialkylamine_((C≤36)),        heterocycloalkane_((C≤12)), or a substituted

    -   version of any of these groups; wherein the repeating unit        comprises a degradable diacyl and a linker;

    -   the degradable diacyl group has the formula:

    -   

    -   wherein:        -   A₁ and A₂ are each independently —O—, —S—, or —NR_(a)—,            wherein:

        -   R_(a) is hydrogen, alkyl_((C≤6)), or substituted            alkyl_((C≤6));

        -   Y₃ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),            arenediyl_((C≤12)), or a substituted version of any of these            groups; or a group of the formula:

        -   

        -   

        -   wherein:

        -   X₃ and X₄ are alkanediyl_((C≤12)), alkenediyl_((C≤12)),            arenediyl_((C≤12)), or a substituted version of any of these            groups;

        -   Y₅ is a covalent bond, alkanediyl_((C≤12)),            alkenediyl_((C≤12)), arenediyl_((C≤12)), or a substituted            version of any of these groups; and

        -   R₉ is alkyl_((C≤8)) or substituted alkyl_((C≤8));

    -   the linker group has the formula:

    -   

    -   wherein:        -   Y₁ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),            arenediyl_((C≤12)), or a substituted version of any of these            groups; and

    -   wherein when the repeating unit comprises a linker group, then        the linker group comprises an independent degradable diacyl        group attached to both the nitrogen and the sulfur atoms of the        linker group if n is greater than 1, wherein the first group in        the repeating unit is a degradable diacyl group, wherein for        each linker group, the next repeating unit comprises two        degradable diacyl groups attached to the nitrogen atom of the        linker group; and wherein n is the number of linker groups        present in the repeating unit; and

-   the terminating group has the formula:

-   

-   wherein:    -   Y₄ is alkanediyl_((C≤18)) or an alkanediyl_((C≤18)) wherein one        or more of the hydrogen atoms on the alkanediyl_((C≤18)) has        been replaced with —OH, —F, —Cl, —Br, —I, —SH, —OCH₃, —OCH₂CH₃,        —SCH₃, or —OC(O)CH₃;    -   R₁₀ is hydrogen, carboxy, hydroxy, or    -   aryl_((C≤12)), alkylamino_((C≤12)), dialkylamino_((C≤12)),        N-heterocycloalkyl_((C≤12)),        —C(O)N(R₁₁)—alkanediyl_((C≤6))-heterocycloalkyl_((C<12)),        —C(O)—alkylamino_((C≤12)), —C(O)—dialkylamino_((C≤12)),        —C(O)—N—heterocycloalkyl_((C≤12)), wherein:    -   R₁₁ is hydrogen, alkyl_((C≤6)), or substituted alkyl_((C≤6));    -   wherein the final degradable diacyl in the chain is attached to        a terminating group;    -   n is 0, 1, 2, 3, 4, 5, or 6;

-   or a pharmaceutically acceptable salt thereof. In some embodiments,    the terminating group is further defined by the formula:

-   

-   wherein:    -   Y₄ is alkanediyl_((C≤18)); and    -   R₁₀ is hydrogen. In some embodiments, A₁ and A₂ are each        independently —O— or —NR_(a)—.

In some embodiments of the dendrimer or dendron of formula (D-I), thecore is further defined by the formula:

wherein:

-   X₂ is N(R₅)_(y);    -   R₅ is hydrogen or alkyl_((C≤8)), or substituted alkyl_((C≤18));        and    -   y is 0, 1, or 2, provided that the sum of y and z is 3;-   R₂ is amino, hydroxy, or mercapto, or alkylamino_((C≤12)),    dialkylamino_((C≤12)), or a substituted version of either of these    groups;-   b is 1, 2, 3, 4, 5, or 6; and-   z is 1, 2, 3; provided that the sum of z and y is 3.

In some embodiments of the dendrimer or dendron of formula (D-I), thecore is further defined by the formula:

wherein:

-   X₃ is —NR₆—, wherein R₆ is hydrogen, alkyl_((C≤8)), or substituted    alkyl_((C≤8)), —O—, or alkylaminodiyl_((C≤8)), alkoxydiyl_((C≤8)),    arenediyl_((C≤8)), heteroarenediyl_((C≤8)),    heterocycloalkanediyl_((C≤8)), or a substituted version of any of    these groups;

-   R₃ and R₄ are each independently amino, hydroxy, or mercapto, or    alkylamino(_(C≤12)), dialkylamino_((C≤12)), or a substituted version    of either of these groups; or a group of the formula:    —N(R_(f))_(f)(CH₂CH₂N(R_(c)))_(e)R_(d),

-   

-   

-   

-   wherein:    -   e and f are each independently 1, 2, or 3; provided that the sum        of e and f is 3;    -   R_(c), R_(d), and R_(f) are each independently hydrogen,        alkyl_((C≤6)), or substituted alkyl_((C≤6));    -   c and d are each independently 1, 2, 3, 4, 5, or 6.

In some embodiments of the dendrimer or dendron of formula (I), theterminating group is represented by the formula:

wherein:

-   Y₄ is alkanediyl_((C≤18)); and-   R₁₀ is hydrogen.

In some embodiments of the dendrimer or dendron of formula (D-I), thecore is further defined as:

In some embodiments of the dendrimer or dendron of formula (D-I), thedegradable diacyl is further defined as:

In some embodiments of the dendrimer or dendron of formula (D-I), thelinker is further defined as

wherein Y₁ is alkanediyl_((C≤8)) or substituted alkanediyl_((C≤8)).

In some embodiments of the dendrimer or dendron of formula (D-I), thedendrimer or dendron is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.

Dendrimers or Dendrons of Formula (X)

In some embodiments of the lipid composition, the ionizable cationiclipid is a dendrimer or dendron of the formula

. In some embodiments, the ionizable cationic lipid is a dendrimer ordendron of the formula

In some embodiments of the lipid composition, the ionizable cationiclipid is a dendrimer or dendron of a generation (g) having a structuralformula:

or a pharmaceutically acceptable salt thereof, wherein:

-   (a) the core comprises a structural formula (X_(Core)):

-   

-   wherein:    -   Q is independently at each occurrence a covalent bond, —O—, —S—,        —NR²—, or —CR^(3a)R^(3b)—;    -   R² is independently at each occurrence R^(1g) or        —L²—NR^(1e)R^(1f);    -   R^(3a) and R^(3b) are each independently at each occurrence        hydrogen or an optionally substituted (e.g., C₁-C₆, such as        C₁-C₃) alkyl;    -   R^(1a), R^(1b), R^(1c), R^(1d), R^(1c), R^(1f), and R^(1g) (if        present) are each independently at each occurrence a point of        connection to a branch, hydrogen, or an optionally substituted        (e.g., C₁-C₁₂) alkyl;    -   L⁰, L¹, and L² are each independently at each occurrence        selected from a covalent bond, alkylene, heteroalkylene,        [alkylene]-[heterocycloalkyl]-[alkylene],        [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene;        or,    -   alternatively, part of L¹ form a (e.g., C₄-C₆) heterocycloalkyl        (e.g., containing one or two nitrogen atoms and, optionally, an        additional heteroatom selected from oxygen and sulfur) with one        of R^(1c) and R^(1d); and    -   x¹ is 0, 1, 2, 3, 4, 5, or 6; and

-   (b) each branch of the plurality (N) of branches independently    comprises a structural formula (X_(Branch)):

-   

-   wherein:    -   * indicates a point of attachment of the branch to the core;    -   g is 1, 2, 3, or 4;    -   Z = 2^((g-1));    -   G=0, when g=1; or    -   $\text{G =}{\sum_{i = 0}^{i = g - 2}2^{\text{i}}},$    -   when g≠1;

-   (c) each diacyl group independently comprises a structural formula

-   

-   wherein:    -   * indicates a point of attachment of the diacyl group at the        proximal end thereof;    -   ** indicates a point of attachment of the diacyl group at the        distal end thereof;    -   Y³ is independently at each occurrence an optionally substituted        (e.g., C₁-C₁₂); alkylene, an optionally substituted (e.g.,        C₁-C₁₂) alkenylene, or an optionally substituted (e.g., C₁-C₁₂)        arenylene;    -   A¹ and A² are each independently at each occurrence —O—, —S—, or        —NR⁴—, wherein:        -   R⁴ is hydrogen or optionally substituted (e.g., C₁-C₆)            alkyl;    -   m¹ and m² are each independently at each occurrence 1, 2, or 3;        and    -   R^(3c), R^(3d), R^(3e), and R^(3f) are each independently at        each occurrence hydrogen or an optionally substituted (e.g.,        C₁-C₈) alkyl; and

-   (d) each linker group independently comprises a structural formula

-   

-   wherein:    -   ** indicates a point of attachment of the linker to a proximal        diacyl group;    -   *** indicates a point of attachment of the linker to a distal        diacyl group; and    -   Y₁ is independently at each occurrence an optionally substituted        (e.g., C₁-C₁₂) alkylene, an optionally substituted (e.g.,        C₁-C₁₂) alkenylene, or an optionally substituted (e.g., C₁-C₁₂)        arenylene; and

-   (e) each terminating group is independently selected from optionally    substituted (e.g., C₁-C₁₈, such as C₄-C₁₈) alkylthiol, and    optionally substituted (e.g., C₁-C₁₈, such as C₄-C₁₈) alkenylthiol.

In some embodiments of X_(Core), Q is independently at each occurrence acovalent bond, —O—, —S—, —NR²—, or —CR^(3a)R^(3b). In some embodimentsof X_(Core) Q is independently at each occurrence a covalent bond. Insome embodiments of X_(Core) Q is independently at each occurrence an—O—. In some embodiments of X_(Core) Q is independently at eachoccurrence a —S—. In some embodiments of X_(Core) Q is independently ateach occurrence a —NR² and R² is independently at each occurrence R^(1g)or —L²—NR^(1e)R^(1f). In some embodiments of X_(Core) Q is independentlyat each occurrence a —CR^(3a)R^(3b) R^(3d), and R^(3a) and R^(3b) areeach independently at each occurrence hydrogen or an optionallysubstituted alkyl (e.g., C₁-C₆, such as C₁-C₃).

In some embodiments of X_(Core), R^(1a), R^(1b), R^(1c), R^(1d), R^(1e),R^(1f), and R^(1g) (if present) are each independently at eachoccurrence a point of connection to a branch, hydrogen, or an optionallysubstituted alkyl. In some embodiments of X_(Core), R^(1a), R^(1b),R^(1c), R^(1d), R^(1e), R^(1f), and R^(1g) (if present) are eachindependently at each occurrence a point of connection to a branch,hydrogen. In some embodiments of X_(Core), R^(1a), R^(1b), R^(1c),R^(1d), R^(1e), R^(1f), and R^(1g) (if present) are each independentlyat each occurrence a point of connection to a branch an optionallysubstituted alkyl (e.g., C₁-C₁₂).

In some embodiments of X_(Core), L⁰, L¹, and L² are each independentlyat each occurrence selected from a covalent bond, alkylene,heteroalkylene, [alkylene]-[heterocycloalkyl]-[alkylene],[alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or,alternatively, part of L¹ form a heterocycloalkyl (e.g., C₄-C₆ andcontaining one or two nitrogen atoms and, optionally, an additionalheteroatom selected from oxygen and sulfur) with one of R^(1c) andR^(1d). In some embodiments of X_(Core), L⁰, L¹, and L² are eachindependently at each occurrence can be a covalent bond. In someembodiments of X_(Core), L⁰, L¹, and L² are each independently at eachoccurrence can be a hydrogen. In some embodiments of X_(Core), L⁰, L¹,and L² are each independently at each occurrence can be an alkylene(e.g., C₁-C₁₂, such as C₁-C₆ or C₁-C₃). In some embodiments of X_(Core),L⁰, L¹, and L² are each independently at each occurrence can be aheteroalkylene (e.g., C₁-C₁₂, such as C₁-C₈ or C₁-C₆). In someembodiments of X_(Core), L⁰, L¹, and L² are each independently at eachoccurrence can be a heteroalkylene (e.g., C₂-C₈ alkyleneoxide, such asoligo(ethyleneoxide)). In some embodiments of X_(Core), L⁰, L¹, and L²are each independently at each occurrence can be a[alkylene]-[heterocycloalkyl]-[alkylene] [(e.g., C₁-C₆)alkylene]-[(e.g., C₄-C₆) heterocycloalkyl]-[(e.g., C₁-C₆) alkylene]. Insome embodiments of X_(Core), L⁰, L¹, and L² are each independently ateach occurrence can be a [alkylene]-(arylene)-[alkylene] [(e.g., C₁-C₆)alkylene]-(arylene)-[(e.g., C₁-C₆) alkylene]. In some embodiments ofX_(Core), L⁰, L¹, and L² are each independently at each occurrence canbe a [alkylene]-(arylene)-[alkylene] (e.g., [(e.g., C₁-C₆)alkylene]-phenylene-[(e.g., C₁-C₆) alkylene]). In some embodiments ofX_(Core), L⁰, L¹, and L² are each independently at each occurrence canbe a heterocycloalkyl (e.g., C₄-C₆heterocycloalkyl). In some embodimentsof X_(Core), L⁰, L¹, and L² are each independently at each occurrencecan be an arylene (e.g., phenylene). In some embodiments of X_(Core),part of L¹ form a heterocycloalkyl with one of R^(1c) and R^(1d). Insome embodiments of X_(Core), part of L¹ form a heterocycloalkyl (e.g.,C₄-C₆ heterocycloalkyl) with one of R^(1c) and R^(1d) and theheterocycloalkyl can contain one or two nitrogen atoms and, optionally,an additional heteroatom selected from oxygen and sulfur.

In some embodiments of X_(Core), L⁰, L¹, and L² are each independentlyat each occurrence selected from a covalent bond, C₁-C₆ alkylene (e.g.,C₁-C₃ alkylene), C₂-C₁₂ (e.g., C₂-C₈) alkyleneoxide (e.g.,oligo(ethyleneoxide), such as -(CH₂CH₂O)₁₋₄—(CH₂CH₂)—), [(C₁-C₄)alkylene]-[(C₄-C₆) heterocycloalkyl]-[(C₁-C₄) alkylene] (e.g.,

and [(C₁-C₄) alkylene]-phenylene-[(C₁-C₄) alkylene] (e.g.,

In some embodiments of X_(Core), L⁰, L¹, and L² are each independentlyat each occurrence selected from C₁-C₆ alkylene (e.g., C₁-C₃ alkylene),-(C₁-C₃ alkylene-O)₁₋₄-(C₁-C₃ alkylene), -(C₁-C₃alkylene)-phenylene-(C₁-C₃ alkylene)-, and -(C₁-C₃alkylene)-piperazinyl-(C₁-C₃ alkylene)-. In some embodiments ofX_(Core), L⁰, L¹, and L² are each independently at each occurrence C₁-C₆alkylene (e.g., C₁-C₃ alkylene). In some embodiments, L⁰, L¹, and L² areeach independently at each occurrence C₂-C₁₂ (e.g., C₂-C₈) alkyleneoxide(e.g., -(C₁-C₃ alkylene-O)₁₋₄-(C₁-C₃ alkylene)). In some embodiments ofX_(Core), L⁰, L¹, and L² are each independently at each occurrenceselected from [(C₁-C₄) alkylene]-[(C₄-C₆) heterocycloalkyl]-[(C₁-C₄)alkylene] (e.g., -(C₁-C₃ alkylene)-phenylene-(C₁-C₃ alkylene)-) and[(C₁-C₄) alkylene]-[(C₄-C₆) heterocycloalkyl]-[(C₁-C₄) alkylene] (e.g.,-(C₁-C₃ alkylene)-piperazinyl-(C₁-C₃ alkylene)-).

In some embodiments of X_(Core), x¹ is 0, 1, 2, 3, 4, 5, or 6. In someembodiments of X_(Core,) x¹ is 0. In some embodiments of X_(Core), x¹is 1. In some embodiments of X_(Core), x¹ is 2. In some embodiments ofX_(Core), x¹ is 0, 3. In some embodiments of X_(Core) x¹ is 4. In someembodiments of X_(Core) x¹ is 5. In some embodiments of X_(Core), x¹ is6.

In some embodiments of X_(Core), the core comprises a structuralformula:

In some embodiments of X_(Core), the core comprises a structuralformula:

In some embodiments of X_(Core), the core comprises a structuralformula:

In some embodiments of X_(Core), the core comprises a structuralformula:

(e.g.,

In some embodiments of X_(Core), the core comprises a structuralformula:

In some embodiments of X_(Core), the core comprises a structuralformula:

In some embodiments of X_(Core), the core comprises a structuralformula:

as

In some embodiments of X_(Core), the core comprises a structuralformula:

wherein Q′ is —NR²— or —CR^(3a)R^(3b)—; q¹ and q² are each independently1 or 2. In some embodiments of X_(Core), the core comprises a structuralformula:

In some embodiments of X_(Core), the core comprises a structural formula

wherein ring A is an optionally substituted aryl or an optionallysubstituted (e.g., C₃-C₁₂, such as C₃-C₅) heteroaryl. In someembodiments of X_(Core), the core comprises has a structural formula

In some embodiments of X_(Core), the core comprises a structural formulaset forth in Table. 1 and pharmaceutically acceptable salts thereof,wherein * indicates a point of attachment of the core to a branch of theplurality of branches. In some embodiments, the example cores of Table.1 are not limited to the stereoisomers (i.e. enantiomers, diastereomers)listed.

TABLE 1 Example core structures ID # Structure 1A1

1A2-1

1A2-2

1A3-1

1A3-2

1A4

1A5-1

1A5-2

2A1-1

2A1-2

2A2-1

2A2-2

2A3

2A4

2A5

2A6

2A7-1

2A7-2

2A8

2A9

2A9V

2A10

2A11

2A12

3A1

3A2

3A3

3A4

3A5

3A6

3A7

4A1

4A2

4A3

4A4

5A1

5A2-1 (5-arm)

5A2-2 (5-arm)

5A2-3 (5-arm)

5A2-4 (5-arm)

5A3-1 (5-arm)

5A4-1 (5-arm)

5A5

5A6

5A2-4 (6 arm)

5A2-5 (6 arm)

5A2-6 (6 arm)

5A3-2 (6 arm)

5A4-2 (6 arm)

6A4

1H1

1H2

1H3

2H1

2H2

2H3

2H4

2H5

2H6

In some embodiments of X_(Core), the core comprises a structural formulaselected from the group consisting of:

pharmaceutically acceptable salts thereof, wherein * indicates a pointof attachment of the core to a branch of the plurality of branches or H.In some embodiments, wherein * indicates a point of attachment of thecore to a branch of the plurality of branches.

In some embodiments of X_(Core), the core has the structure

wherein * indicates a point of attachment of the core to a branch of theplurality of branches or H. In some embodiments, at least 2 branches areattached to the core. In some embodiments, at least 3 branches areattached to the core. In some embodiments, at least 4 branches areattached to the core.

In some embodiments of X_(Core), the core has the structure

wherein * indicates a point of attachment of the core to a branch of theplurality of branches or H. In some embodiments, at least 4 branches areattached to the core. In some embodiments, at least 5 branches areattached to the core. In some embodiments, at least 6 branches areattached to the core.

In some embodiments, the plurality (N) of branches comprises at least 3branches, at least 4 branches, at least 5 branches. In some embodiments,the plurality (N) of branches comprises at least 3 branches. In someembodiments, the plurality (N) of branches comprises at least 4branches. In some embodiments, the plurality (N) of branches comprisesat least 5 branches.

In some embodiments of X_(Branch), g is 1, 2, 3, or 4. In someembodiments of X_(Branch), g is 1. In some embodiments of X_(Branch), gis 2. In some embodiments of X_(Branch), g is 3. In some embodiments ofX_(Branch), g is 4.

In some embodiments of X_(Branch), Z = 2^((g-1)) and when g=1, G=0. Insome embodiments of X_(Branch), Z = 2^((g-1)) and

$\text{G =}{\sum_{i = 0}^{i = g - 2}2^{\text{i}}},$

when g≠1.

In some embodiments of X_(Branch), g=1, G=0, Z=1, and each branch of theplurality of branches comprises a structural formula each branch of theplurality of branches comprises a structural formula

In some embodiments of X_(Branch), g=2, G=1, Z=2, and each branch of theplurality of branches comprises a structural formula

In some embodiments of X_(Branch), g=3, G=3, Z=4, and each branch of theplurality of branches comprises a structural formula

In some embodiments of X_(Branch), g=4, G=7, Z=8, and each branch of theplurality of branches comprises a structural formula

In some embodiments, the dendrimers or dendrons described herein with ageneration

(g) = 1 has the structure: .

In some embodiments, the dendrimers or dendrons described herein with ageneration (g) = 1 has the structure:

An example formulation of the dendrimers or dendrons described hereinfor generations 1-4 is shown in Table 2. The number of diacyl groups,linker groups, and terminating groups can be calculated based on g.

TABLE 2 Formulation of Dendrimer or Dendron Groups Based on Generation(g) g = 1 g = 2 g = 3 g = 4 # of diacyl grp 1 1+2=3 1+2+2²=71+2+2²+2³=15 1+2+...+2^(g-1) # of linker grp 0 1 1+2 1+2+2²1+2+...+2^(g-2) # of terminating grp 1 2 2² 2³ 2^((g-1))

In some embodiments, the diacyl group independently comprises astructural formula

* indicates a point of attachment of the diacyl group at the proximalend thereof, and ** indicates a point of attachment of the diacyl groupat the distal end thereof.

In some embodiments of the diacyl group of X_(Branch), Y³ isindependently at each occurrence an optionally substituted; alkylene, anoptionally substituted alkenylene, or an optionally substitutedarenylene. In some embodiments of the diacyl group of X_(Branch), Y³ isindependently at each occurrence an optionally substituted alkylene(e.g., C₁-C₁₂). In some embodiments of the diacyl group of X_(Branch),Y³ is independently at each occurrence an optionally substitutedalkenylene (e.g., C₁-C₁₂). In some embodiments of the diacyl group ofX_(Branch), Y³ is independently at each occurrence an optionallysubstituted arenylene (e.g., C₁-C₁₂).

In some embodiments of the diacyl group of X_(Branch), A¹ and A ² areeach independently at each occurrence —O—, —S—, or —NR⁴—. In someembodiments of the diacyl group of X_(Branch), A¹ and A² are eachindependently at each occurrence —O—. In some embodiments of the diacylgroup of X_(Branch), A¹ and A² are each independently at each occurrence—S—. In some embodiments of the diacyl group of X_(Branch), A¹ and A²are each independently at each occurrence —NR⁴— and R⁴ is hydrogen oroptionally substituted alkyl (e.g., C₁-C₆). In some embodiments of thediacyl group of X_(Branch), m¹ and m² are each independently at eachoccurrence 1, 2, or 3. In some embodiments of the diacyl group ofX_(Branch), m¹ and m² are each independently at each occurrence 1. Insome embodiments of the diacyl group of X_(Branch), m¹ and m² are eachindependently at each occurrence 2. In some embodiments of the diacylgroup of X_(Branch), m¹ and m² are each independently at each occurrence3. In some embodiments of the diacyl group of X_(Branch), R^(3c),R^(3d), R^(3e), and R^(3f) are each independently at each occurrencehydrogen or an optionally substituted alkyl. In some embodiments of thediacyl group of X_(Branch), R^(3c), R^(3d), R^(3e), and R^(3f) are eachindependently at each occurrence hydrogen. In some embodiments of thediacyl group of X_(Branch), R^(3c), R^(3d), R^(3e), and R^(3f) are eachindependently at each occurrence an optionally substituted (e.g., C₁-C₈)alkyl.

In some embodiments of the diacyl group, A¹ is —O— or —NH—. In someembodiments of the diacyl group, A¹ is —O—. In some embodiments of thediacyl group, A² is —O— or —NH—. In some embodiments of the diacylgroup, A² is —O—. In some embodiments of the diacyl group, Y³ is C₁-C₁₂(e.g., C₁-C₆, such as C₁-C₃) alkylene.

In some embodiments of the diacyl group, the diacyl group independentlyat each occurrence comprises a structural formula

(e.g.,

such as

and optionally R^(3c), R^(3d), R^(3e), and R^(3f) are each independentlyat each occurrence hydrogen or C₁-C₃ alkyl.

In some embodiments, linker group independently comprises a structuralformula

** indicates a point of attachment of the linker to a proximal diacylgroup, and *** indicates a point of attachment of the linker to a distaldiacyl group.

In some embodiments of the linker group of X_(Branch) if present, Y₁ isindependently at each occurrence an optionally substituted alkylene, anoptionally substituted alkenylene, or an optionally substitutedarenylene. In some embodiments of the linker group of X_(Branch) ifpresent, Y₁ is independently at each occurrence an optionallysubstituted alkylene (e.g., C₁-C₁₂). In some embodiments of the linkergroup of X_(Branch) if present, Y₁ is independently at each occurrencean optionally substituted alkenylene (e.g., C₁-C₁₂). In some embodimentsof the linker group of X_(Branch) if present, Y₁ is independently ateach occurrence an optionally substituted arenylene (e.g., C₁-C₁₂).

In some embodiments of the terminating group of X_(Branch), eachterminating group is independently selected from optionally substitutedalkylthiol and optionally substituted alkenylthiol. In some embodimentsof the terminating group of X_(Branch), each terminating group is anoptionally substituted alkylthiol (e.g., C₁-C₁₈, such as C₄-C₁₈). Insome embodiments of the terminating group of X_(Branch), eachterminating group is optionally substituted alkenylthiol (e.g., C₁-C₁₈,such as C₄-C₁₈).

In some embodiments of the terminating group of X_(Branch), eachterminating group is independently C₁-C₁₈ alkenylthiol or C₁-C₁₈alkylthiol, and the alkyl or alkenyl moiety is optionally substitutedwith one or more substituents each independently selected from halogen,C₆-C₁₂ aryl, C₁-C₁₂ alkylamino, C₄-C₆ N-heterocycloalkyl, —OH, —C(O)OH,—C(O)N(C₁-C₃ alkyl)-(C₁-C₆ alkylene)-(C₁-C₁₂ alkylamino), —C(O)N(C₁-C₃alkyl)-(C₁-C₆ alkylene)-(C₄-C₆ N-heterocycloalkyl), —C(O)—(C₁-C₁₂alkylamino), and —C(O)—(C₄-C₆ N-heterocycloalkyl), and the C₄-C₆N-heterocycloalkyl moiety of any of the preceding substituents isoptionally substituted with C₁-C₃ alkyl or C₁-C₃ hydroxyalkyl.

In some embodiments of the terminating group of X_(Branch), eachterminating group is independently C₁-C₁₈ (e.g., C₄-C₁₈) alkenylthiol orC₁-C₁₈ (e.g., C₄-C₁₈) alkylthiol, wherein the alkyl or alkenyl moiety isoptionally substituted with one or more substituents each independentlyselected from halogen, C₆-C₁₂ aryl (e.g., phenyl), C₁-C₁₂ (e.g., C₁-C₈)alkylamino (e.g., C₁-C₆ mono-alkylamino (such as —NHCH₂CH₂CH₂CH₃) orC₁-C₈ di-alkylamino (such as

C₄-C₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl (

N-piperidinyl

N-azepanyl

—OH, —C(O)OH, —C(O)N(C₁-C₃ alkyl)-(C₁-C₆ alkylene)-(C₁-C₁₂ alkylamino(e.g., mono- or di-alkylamino)) (e.g.,

—C(O)N(C₁-C₃ alkyl)-(C₁-C₆ alkylene)-(C₄-C₆ N-heterocycloalkyl) (e.g.,

—C(O)—(C₁-C₁₂ alkylamino (e.g., mono- or di-alkylamino)), and—C(O)—(C₄-C₆ N-heterocycloalkyl) (e.g.,

wherein the C₄-C₆ N-heterocycloalkyl moiety of any of the precedingsubstituents is optionally substituted with C₁-C₃ alkyl or C₁-C₃hydroxyalkyl. In some embodiments of the terminating group ofX_(Branch), each terminating group is independently C₁-C₁₈ (e.g.,C₄-C₁₈) alkylthiol, wherein the alkyl moiety is optionally substitutedwith one substituent —OH. In some embodiments of the terminating groupof X_(Branch), each terminating group is independently C₁-C₁₈ (e.g.,C₄-C₁₈) alkylthiol, wherein the alkyl moiety is optionally substitutedwith one substituent selected from C₁-C₁₂ (e.g., C₁-C₈) alkylamino(e.g., C₁-C₆ mono-alkylamino (such as —NHCH₂CH₂CH₂CH₃) or C₁-C₈di-alkylamino (such as

and C₄-C₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl

N-piperidinyl

N-azepanyl

In some embodiments of the terminating group of X_(Branch), eachterminating group is independently C₁-C₁₈ (e.g., C₄-C₁₈) alkenylthiol orC₁-C₁₈ (e.g., C₄-C₁₈) alkylthiol. In some embodiments of the terminatinggroup of X_(Branch), each terminating group is independently C₁-C₁₈(e.g., C₄-C₁₈) alkylthiol.

In some embodiments of the terminating group of X_(Branch), eachterminating group is independently a structural set forth in Table 3. Insome embodiments, the dendrimers or dendrons described herein cancomprise a terminating group or pharmaceutically acceptable salt, orthereof selected in Table 3. In some embodiments, the exampleterminating group of Table 3 are not limiting of the stereoisomers (i.e.enantiomers, diastereomers) listed.

TABLE 3 Example terminating group / peripheries structures ID #Structure SC1

SC2

SC3

SC4

SC5

SC6

SC7

SC8

SC9

SC10

SC11

SC12

SC14

SC16

SC18

SC19

SO1

SO2

SO3

SO4

SO5

SO6

SO7

SO8

S09

SN1

SN2

SN3

SN4

SN5

SN6

SN7

SN8

SN9

SN10

SN11

In some embodiments, the dendrimer or dendron of Formula (X) is selectedfrom those set forth in Table 4 and pharmaceutically acceptable saltsthereof.

TABLE 4 Example ionizable cationic lipo-dendrimers or lipo-dendrons ID #Structure 2A2-SC14

2A6-SC14

2A9-SC14

3A3-SC10

3A3-SC14

3A5-SC10

3A5-SC14

4A1-SC12

4A3-SC12

5A1-SC12

5A1-SC8

5A2-2-SC12 (5-arm)

5A3-1-SC12 (5 arm)

5A3-1-SC8 (5-arm)

5A4-1-SC12 (5-arm)

5A4-1-SC8 (5-arm)

5A5-SC8

5A5-SC12

5A2-4-SC12 (6-arm)

5A2-4-SC10 (6-arm)

5A3-2--SC8 (6-arm)

5A3-2-SC12 (6-arm)

5A4-2-SC8 (6-arm)

5A4-2-SC12 (6-arm)

6A4-SC8

6A4-SC12

2A2-g2-SC12

2A2-g2-SC8

2A11-g2-SC12

2A11-g2-SC8

3A3-g2-SC12

3A3-g2-SC8

3A5-g2-SC12

2A11-g3-SC12

2A11-g3-SC8

1A2-g4-SC12

4A1-g2-SC12

1A2-g4-SC8

4A1-g2-SC8

4A3-g2-SC12

4A3-g2-SC8

1A2-g3-SC12

1A2-g3-SC8

2A2-g3-SC12

2A2-g3-SC8

5A2-4-SC8 (6-arm)

5A-5-SC8 (6 arm)

5A2-6-SC8 (6 arm)

5A2-1-SC8 (5-arm)

5A2-2-SC8

4A1-SC5

4A1-SC8

4A3-SC6

4A3-SC7

4A3-SC8

5A4-2-SC5 (6 arm)

5A4-2-SC6 (6 arm)

5A2-4-SC8 (5-arm)

3A5-g2-SC8

Other Ionizable Cationic Lipids

In some embodiments of the lipid composition, the cationic lipidcomprises a structural formula (D-I′):

wherein:

-   a is 1 and b is 2, 3, or 4; or, alternatively, b is 1 and a is 2, 3,    or 4;-   m is 1 and n is 1; or, alternatively, m is 2 and n is 0; or,    alternatively, m is 2 and n is 1; and-   R¹, R², R³, R⁴, R⁵, and R⁶ are each independently selected from the    group consisting of H, —CH₂CH(OH)R⁷, —CH(R⁷)CH₂OH, —CH₂CH₂C(═O)OR ⁷,    —CH₂CH₂C(═O)NHR⁷, and —CH₂R⁷, wherein R⁷ is independently selected    from C₃-C₁₈ alkyl, C₃-C₁₈ alkenyl having one C═C double bond, a    protecting group for an amino group, —C(═NH)NH₂, a poly(ethylene    glycol) chain, and a receptor ligand;-   provided that at least two moieties among R¹ to R⁶ are independently    selected from —CH₂CH(OH)R⁷, —CH(R⁷)CH₂OH, —CH₂CH₂C(═O)OR⁷,    —CH₂CH₂C(═O)NHR⁷, or —CH₂R⁷, wherein R⁷ is independently selected    from C₃-C₁₈ alkyl or C₃-C₁₈ alkenyl having one C=C double bond; and-   wherein one or more of the nitrogen atoms indicated in formula    (D-I′) may be protonated to provide a cationic lipid.

In some embodiments of the cationic lipid of formula (D-I′), a is 1. Insome embodiments of the cationic lipid of formula (D-I′), b is 2. Insome embodiments of the cationic lipid of formula (D-I′), m is 1. Insome embodiments of the cationic lipid of formula (D-I′), n is 1. Insome embodiments of the cationic lipid of formula (D-I′), R¹, R², R³,R⁴, R⁵, and R⁶ are each independently H or —CH₂CH(OH)R⁷. In someembodiments of the cationic lipid of formula (_(D-I)′), R¹, R², R³, R⁴,R⁵, and R⁶ are each independently H or

In some embodiments of the cationic lipid of formula (D-I′), R¹, R², R³,R⁴, R⁵, and R⁶ are each independently H or

In some embodiments of the cationic lipid of formula (D-I′), R⁷ isC₃-C₁₈ alkyl (e.g., C₆-C₁₂ alkyl).

In some embodiments, the cationic lipid of formula (D-I′) is13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol:

In some embodiments, the cationic lipid of formula (D-I′) is(11R,25R)-13,16,20-tris((R)-2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol:

Additional cationic lipids that can be used in the compositions andmethods of the present application include those cationic lipids asdescribed in J. McClellan, M. C. King, Cell 2010, 141, 210-217, andInternational Patent Publication WO 2010/144740, WO 2013/149140, WO2016/118725, WO 2016/118724, WO 2013/063468, WO 2016/205691, WO2015/184256, WO 2016/004202, WO 2015/199952, WO 2017/004143, WO2017/075531, WO 2017/117528, WO 2017/049245, WO 2017/173054 and WO2015/095340, which are incorporated herein by reference for allpurposes. Examples of those ionizable cationic lipids include but arenot limited to those as shown in Table 5.

TABLE 5 Example ionizable cationic lipids # Structure of exampleionizable cationic lipid 1

2

3

4

5

6

7

8

9

1 0

1 1

1 2

1 3

1 4

1 5

1 6

1 7

1 8

1 9

2 0

2 1

2 2

2 3

2 4

2 5

2 6

2 7

2 8

2 9

3 0

3 1

3 2

3 3

3 4

3 5

3 6

3 7

3 8

3 9

4 0

4 1

4 2

4 3

4 4

4 5

4 6

4 7

4 8

4 9

5 0

5 1

5 2

5 3

5 4

5 5

5 6

5 7

5 8

5 9

6 0

6 1

6 2

6 3

6 4

6 5

6 6

6 7

6 8

6 9

7 0

7 1

7 2

7 3

7 4

7 5

7 6

In some embodiments of the lipid composition of the present application,the ionizable cationic lipid is present in an amount from about fromabout 20 to about 23. In some embodiments, the molar percentage is fromabout 20, 20.5, 21, 21.5, 22, 22.5, to about 23 or any range derivabletherein. In other embodiments, the molar percentage is from about 7.5 toabout 20. In some embodiments, the molar percentage is from about 7.5,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, to about 20 or any rangederivable therein.

In some embodiments of the lipid composition of the present application,the lipid composition comprises the ionizable cationic lipid at a molarpercentage from about 5% to about 30%. In some embodiments of the lipidcomposition of the present application, the lipid composition comprisesthe ionizable cationic lipid at a molar percentage from about 10% toabout 25%. In some embodiments of the lipid composition of the presentapplication, the lipid composition comprises the ionizable cationiclipid at a molar percentage from about 15% to about 20%. In someembodiments of the lipid composition of the present application, thelipid composition comprises the ionizable cationic lipid at a molarpercentage from about 10% to about 20%. In some embodiments of the lipidcomposition of the present application, the lipid composition comprisesthe ionizable cationic lipid at a molar percentage from about 20% toabout 30%. In some embodiments of the lipid composition of the presentapplication, the lipid composition comprises the ionizable cationiclipid at a molar percentage of at least (about) 5%, at least (about)10%, at least (about) 15%, at least (about) 20%, at least (about) 25%,or at least (about) 30%. In some embodiments of the lipid composition ofthe present application, the lipid composition comprises the ionizablecationic lipid at a molar percentage of at most (about) 5%, at most(about) 10%, at most (about) 15%, at most (about) 20%, at most (about)25%, or at most (about) 30%.

Selective Organ Targeting (SORT) Lipids

In some embodiments of the lipid composition of the present application,the lipid (e.g., nanoparticle) composition is preferentially deliveredto a target organ. In some embodiments, the target organ is a lung, alung tissue or a lung cell. As used herein, the term “preferentiallydelivered” is used to refer to a composition, upon being delivered,which is delivered to the target organ (e.g., lung), tissue, or cell inat least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, or 75%) of the amount administered.

In some embodiments of the lipid composition, the lipid compositioncomprises one or more selective organ targeting (SORT) lipid which leadsto the selective delivery of the composition to a particular organ. Insome embodiments, the SORT lipid may have two or more alkyl or alkenylchains of C₆-C₂₄.

In some embodiments of the lipid compositions, the SORT lipid comprisespermanently positively charged moiety. The permanently positivelycharged moiety may be positively charged at a physiological pH such thatthe SORT lipid comprises a positive charge upon delivery of apolynucleotide to a cell. In some embodiments the positively chargedmoiety is quaternary amine or quaternary ammonium ion. In someembodiments, the SORT lipid comprises, or is otherwise complexed to orinteracting with, a counterion.

In some embodiments of the lipid compositions, the SORT lipid is apermanently cationic lipid (i.e., comprising one or more hydrophobiccomponents and a permanently cationic group). The permanently cationiclipid may contain a group which has a positive charge regardless of thepH. One permanently cationic group that may be used in the permanentlycationic lipid is a quaternary ammonium group. The permanently cationiclipid may comprise a structural formula:

wherein:

-   Y₁, Y₂, or Y₃ are each independently X₁C(O)R₁ or X₂N⁺R₃R₄R₅;-   provided at least one of Y₁, Y₂, and Y₃ is X₂N⁺R₃R₄R₅;-   R¹ is C₁-C₂₄ alkyl, C₁-C₂₄ substituted alkyl, C₁-C₂₄ alkenyl, C₁-C₂₄    substituted alkenyl;-   X₁ is O or NR_(a), wherein R_(a) is hydrogen, C₁-C₄ alkyl, or C₁-C₄    substituted alkyl;-   X₂ is C₁-C₆ alkanediyl or C₁-C₆ substituted alkanediyl;-   R₃, R₄, and R₅ are each independently C₁-C₂₄ alkyl, C₁-C₂₄    substituted alkyl, C₁-C₂₄ alkenyl, C₁-C₂₄ substituted alkenyl; and-   A₁ is an anion with a charge equal to the number of X₂N⁺R₃R₄R₅    groups in the compound.

In some embodiments of the SORT lipids, the permanently cationic SORTlipid has a structural formula:

wherein:

-   R₆—R₉ are each independently C₁-C₂₄ alkyl, C₁-C₂₄ substituted alkyl,    C₁-C₂₄ alkenyl, C₁-C₂₄ substituted alkenyl; provided at least one of    R₆—R₉ is a group of C₈-C₂₄; and-   A₂ is a monovalent anion.

In some embodiments of the lipid compositions, the SORT lipid isionizable cationic lipid (i.e., comprising one or more hydrophobiccomponents and an ionizable cationic group). The ionizable positivelycharged moiety may be positively charged at a physiological pH. Oneionizable cationic group that may be used in the ionizable cationiclipid is a tertiary ammine group. In some embodiments of the lipidcompositions, the SORT lipid has a structural formula:

wherein:

-   R₁ and R₂ are each independently alkyl_((C8-C24)),    alkenyl_((C8-C24)), or a substituted version of either group; and-   R₃ and R₃′ are each independently alkyl_((C≤6)) or substituted    alkyl_((C≤6)).

In some embodiments of the lipid compositions, the SORT lipid comprisesa head group of a particular structure. In some embodiments, the SORTlipid comprises a headgroup having a structural formula:

wherein L is a linker; Z⁺ is positively charged moiety and X⁻ is acounterion. In some embodiment, the linker is a biodegradable linker.The biodegradable linker may be degradable under physiological pH andtemperature. The biodegradable linker may be degraded by proteins orenzymes from a subject. In some embodiments, the positively chargedmoiety is a quaternary ammonium ion or quaternary amine.

In some embodiments of the lipid compositions, the SORT lipid has astructural formula:

wherein R¹ and R² are each independently an optionally substitutedC₆-C₂₄ alkyl, or an optionally substituted C₆-C₂₄ alkenyl.

In some embodiments of the lipid compositions, the SORT lipid has astructural formula:

In some embodiments of the lipid compositions, the SORT lipid comprisesa Linker (L). In some embodiments, L is

wherein:

-   p and q are each independently 1, 2, or 3; and-   R⁴ is an optionally substituted C₁-C₆ alkyl

In some embodiments of the lipid compositions, the SORT lipid has astructural formula:

wherein:

-   R₁ and R₂ are each independently alkyl_((C8-C24)),    alkenyl_((C8-C24)), or a substituted version of either group;-   R₃, R₃′, and R₃“ are each independently alkyl_((C≤6)) or substituted    alkyl_((C≤6));-   R₄ is alkyl_((C≤6)) or substituted alkyl_((C≤6)); and-   X⁻ is a monovalent anion.

In some embodiments of the lipid compositions, the SORT lipid is aphosphatidylcholine (e.g., 14:0 EPC). In some embodiments, thephosphatidylcholine compound is further defined as:

wherein:

-   R₁ and R₂ are each independently alkyl_((C8-C24)),    alkenyl_((C8-C24)), or a substituted version of either group;-   R₃, R₃′, and R₃“ are each independently alkyl_((C≤6)) or substituted    alkyl_((C≤6)); and-   X⁻ is a monovalent anion.

In some embodiments of the lipid compositions, the SORT lipid is aphosphocholine lipid. In some embodiments, the SORT lipid is anethylphosphocholine. The ethylphosphocholine may be, by way of example,without being limited to,1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine,1,2-dioleoyl-sn-glycero-3-ethylphosphocholine,1,2-distearoyl-sn-glycero-3-ethylphosphocholine,1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine,1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine,1,2-dilauroyl-sn-glycero-3-ethylphosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine.

In some embodiments of the lipid compositions, the SORT lipid has astructural formula:

wherein:

-   R₁ and R₂ are each independently alkyl_((C8-C24)),    alkenyl_((C8-C24)), or a substituted version of either group; R₃,    R₃′, and R₃“ are each independently alkyl_((C≤6)) or substituted    alkyl_((C≤6));-   X⁻ is a monovalent anion.

By way of example, and without being limited thereto, a SORT lipid ofthe structural formula of the immediately preceding paragraph is1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., chloridesalt).

In some embodiments of the lipid compositions, the SORT lipid has astructural formula:

wherein:

-   R₄ and R₄′ are each independently alkyl_((C8-C24)),    alkenyl_((C6-C24)), or a substituted version of either group;-   R₄“ is alkyl_((C≤24)), alkenyl_((C≤24)), or a substituted version of    either group;-   R₄′“ is alkyl_((C1-C8)), alkenyl_((C2-C8)), or a substituted version    of either group; and-   X₂ is a monovalent anion.

By way of example, and without being limited thereto, a SORT lipid ofthe structural formula of the immediately preceding paragraph isdimethyldioctadecylammonium (DDAB) (e.g., bromide salt).

In some embodiments of the lipid compositions, the SORT lipid has astructural formula:

wherein:

-   R₁ and R₂ are each independently alkyl_((C8-C24)),    alkenyl_((C8-C24)), or a substituted version of either group;-   R₃, R₃′, and R₃“ are each independently alkyl_((C≤6)) or substituted    alkyl_((C≤6)); and-   X⁻ is a monovalent anion.

By way of example, and without being limited thereto, a SORT lipid ofthe structural formula of the immediately preceding paragraph isN—[1-(2, 3-dioleyloxy)propyl]—N,N,N-trimethylammonium chloride (DOTMA).

In some embodiments of the lipid compositions, the SORT lipid is ananionic lipid. In some embodiments of the lipid compositions, the SORTlipid has a structural formula:

wherein:

-   R₁ and R₂ are each independently alkyl_((C8-C24)),    alkenyl_((C8-C24)), or a substituted version of either group;-   R₃ is hydrogen, alkyl_((C≤6)), or substituted alkyl_((C≤6)), or    —Y₁—R₄, wherein: Y₁ is alkanediyl_((C≤6)) or substituted    alkanediyl_((C≤6)); and-   R₄ is acyloxy_((C≤8-24)) or substituted acyloxy_((C≤8-24)).

In some embodiments of the lipid compositions, the SORT lipid comprisesone or more selected from the lipids set forth in Table 6.

TABLE 6 Example SORT lipids Lipid Name Structure1,2-Dioleoyl-3-dimethylammonium-propane (18:1 DODAP)

1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP) (e.g., chloridesalt)

1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP) (e.g., chloridesalt)

1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP) (e.g., chloridesalt)

1,2-Dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., chloridesalt)

1,2-Di-O-octadecenyl-3-trimethylammonium propane (DOTMA) (e.g., chloridesalt)

Dimethyldioctadecylammoniu m (DDAB) (e.g., bromide salt)

1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EPC) (e.g.,chloride salt)

1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC) (e.g., chloridesalt)

1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 EPC) (e.g.,triflate salt)

1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC) (e.g.,chloride salt)

1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EPC) (e.g.,chloride salt)

1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC) (e.g., chloridesalt)

1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 EPC)(e.g., chloride salt)

1,2-di-O-octadecenyl-3-trimethylammonium propane (18:1 DOTMA) (e.g.,chloride salt)

X⁻ is a counterion (e.g., Cl⁻, Br⁻, etc.)

In some embodiments of the lipid composition of the present application,the lipid composition comprises the SORT lipid at a molar percentagefrom about 20% to about 65%. In some embodiments of the lipidcomposition of the present application, the lipid composition comprisesthe SORT lipid at a molar percentage from about 25% to about 60%. Insome embodiments of the lipid composition of the present application,the lipid composition comprises the SORT lipid at a molar percentagefrom about 30% to about 55%. In some embodiments of the lipidcomposition of the present application, the lipid composition comprisesthe SORT lipid at a molar percentage from about 20% to about 50%. Insome embodiments of the lipid composition of the present application,the lipid composition comprises the SORT lipid at a molar percentagefrom about 30% to about 60%. In some embodiments of the lipidcomposition of the present application, the lipid composition comprisesthe SORT lipid at a molar percentage from about 25% to about 60%. Insome embodiments of the lipid composition of the present application,the lipid composition comprises the SORT lipid at a molar percentage ofat least (about) 25%, at least (about) 30%, at least (about) 35%, atleast (about) 40%, at least (about) 45%, at least (about) 50%, at least(about) 55%, at least (about) 60%, or at least (about) 65%. In someembodiments of the lipid composition of the present application, thelipid composition comprises the SORT lipid at a molar percentage of atmost (about) 25%, at most (about) 30%, at most (about) 35%, at most(about) 40%, at least (about) 45%, at most (about) 50%, at most (about)55%, at most (about) 60%, or at most (about) 65%. In some embodiments ofthe lipid composition of the present application, the lipid compositioncomprises the SORT lipid at a molar percentage of about 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, or 65%, or of a range between (inclusive)any two of the foregoing values.

Additional Lipids

In some embodiments of the lipid composition of the present application,the lipid composition further comprises an additional lipid includingbut not limited to a steroid or a steroid derivative, a PEG lipid, and aphospholipid.

Phospholipids or Other Zwitterionic Lipids

In some embodiments of the lipid composition of the present application,the lipid composition further comprises a phospholipid. In someembodiments, the phospholipid may contain one or two long chain (e.g.,C₆-C₂₄) alkyl or alkenyl groups, a glycerol or a sphingosine, one or twophosphate groups, and, optionally, a small organic molecule. The smallorganic molecule may be an amino acid, a sugar, or an amino substitutedalkoxy group, such as choline or ethanolamine. In some embodiments, thephospholipid is a phosphatidylcholine. In some embodiments, thephospholipid is distearoylphosphatidylcholine ordioleoylphosphatidylethanolamine. In some embodiments, otherzwitterionic lipids are used, where zwitterionic lipid defines lipid andlipid-like molecules with both a positive charge and a negative charge.

In some embodiments of the lipid compositions, the phospholipid is notan ethylphosphocholine.

In some embodiments of the lipid composition of the present application,the compositions may further comprise a molar percentage of thephospholipid to the total lipid composition from about 20 to about 23.In some embodiments, the molar percentage is from about 20, 20.5, 21,21.5, 22, 22.5, to about 23 or any range derivable therein. In otherembodiments, the molar percentage is from about 7.5 to about 60. In someembodiments, the molar percentage is from about 7.5, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, to about 20 or any range derivable therein.

In some embodiments of the lipid composition of the present application,the lipid composition comprises the phospholipid at a molar percentagefrom about 8% to about 23%. In some embodiments of the lipid compositionof the present application, the lipid composition comprises thephospholipid at a molar percentage from about 10% to about 20%. In someembodiments of the lipid composition of the present application, thelipid composition comprises the phospholipid at a molar percentage fromabout 15% to about 20%. In some embodiments of the lipid composition ofthe present application, the lipid composition comprises thephospholipid at a molar percentage from about 8% to about 15%. In someembodiments of the lipid composition of the present application, thelipid composition comprises the phospholipid at a molar percentage fromabout 10% to about 15%. In some embodiments of the lipid composition ofthe present application, the lipid composition comprises thephospholipid at a molar percentage from about 12% to about 18%. In someembodiments of the lipid composition of the present application, thelipid composition comprises the phospholipid at a molar percentage of atleast (about) 8%, at least (about) 10%, at least (about) 12%, at least(about) 15%, at least (about) 18%, at least (about) 20%, or at least(about) 23%. In some embodiments of the lipid composition of the presentapplication, the lipid composition comprises the phospholipid at a molarpercentage of at most (about) 8%, at most (about) 10%, at most (about)12%, at most (about) 15%, at most (about) 18%, at most (about) 20%, orat most (about) 23%.

Steroids or Steroid Derivatives

In some embodiments of the lipid composition of the present application,the lipid composition further comprises a steroid or steroid derivative.In some embodiments, the steroid or steroid derivative comprises anysteroid or steroid derivative. As used herein, in some embodiments, theterm “steroid” is a class of compounds with a four ring 17 carbon cyclicstructure which can further comprises one or more substitutionsincluding alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acylgroups, or a double bond between two or more carbon atoms. In oneaspect, the ring structure of a steroid comprises three fused cyclohexylrings and a fused cyclopentyl ring as shown in the formula:

In some embodiments, a steroid derivative comprises the ring structureabove with one or more non-alkyl substitutions. In some embodiments, thesteroid or steroid derivative is a sterol wherein the formula is furtherdefined as:

. In some embodiments of the present application, the steroidor steroidderivative is a cholestane or cholestane derivative. In a cholestane,the ring structure is further defined by the formula:

As described above, a cholestane derivative includes one or morenon-alkyl substitution of the above ring system. In some embodiments,the cholestane or cholestane derivative is a cholestene or cholestenederivative or a sterol or a sterol derivative. In other embodiments, thecholestane or cholestane derivative is both a cholestere and a sterol ora derivative thereof.

In some embodiments of the lipid composition, the compositions mayfurther comprise a molar percentage of the steroid to the total lipidcomposition from about 40 to about 46. In some embodiments, the molarpercentage is from about 40, 41, 42, 43, 44, 45, to about 46 or anyrange derivable therein. In other embodiments, the molar percentage ofthe steroid relative to the total lipid composition is from about 15 toabout 40. In some embodiments, the molar percentage is 15, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or any range derivabletherein.

In some embodiments of the lipid composition of the present application,the lipid composition comprises the steroid or steroid derivative at amolar percentage from about 15% to about 46%. In some embodiments of thelipid composition of the present application, the lipid compositioncomprises the steroid or steroid derivative at a molar percentage fromabout 20% to about 40%. In some embodiments of the lipid composition ofthe present application, the lipid composition comprises the steroid orsteroid derivative at a molar percentage from about 25% to about 35%. Insome embodiments of the lipid composition of the present application,the lipid composition comprises the steroid or steroid derivative at amolar percentage from about 30% to about 40%. In some embodiments of thelipid composition of the present application, the lipid compositioncomprises the steroid or steroid derivative at a molar percentage fromabout 20% to about 30%. In some embodiments of the lipid composition ofthe present application, the lipid composition comprises the steroid orsteroid derivative at a molar percentage of at least (about) 15%, of atleast (about) 20%, of at least (about) 25%, of at least (about) 30%, ofat least (about) 35%, of at least (about) 40%, of at least (about) 45%,or of at least (about) 46%. In some embodiments of the lipid compositionof the present application, the lipid composition comprises the steroidor steroid derivative at a molar percentage of at most (about) 15%, ofat most (about) 20%, of at most (about) 25%, of at most (about) 30%, ofat most (about) 35%, of at most (about) 40%, of at most (about) 45%, orof at most (about) 46%.

Polymer-Conjugated Lipids

In some embodiments of the lipid composition of the present application,the lipid composition further comprises a polymer conjugated lipid. Insome embodiments, the polymer conjugated lipid is a PEG lipid. In someembodiments, the PEG lipid is a diglyceride which also comprises a PEGchain attached to the glycerol group. In other embodiments, the PEGlipid is a compound which contains one or more C₆-C₂₄ long chain alkylor alkenyl group or a C₆-C₂₄ fatty acid group attached to a linker groupwith a PEG chain. Some non-limiting examples of a PEG lipid includes aPEG modified phosphatidylethanolamine and phosphatidic acid, a PEGceramide conjugated, PEG modified dialkylamines and PEG modified1,2-diacyloxypropan-3-amines, PEG modified diacylglycerols anddialkylglycerols. In some embodiments, PEG modifieddiastearoylphosphatidylethanolamine or PEG modifieddimyristoyl-sn-glycerol. In some embodiments, the PEG modification ismeasured by the molecular weight of PEG component of the lipid. In someembodiments, the PEG modification has a molecular weight from about 100to about 15,000. In some embodiments, the molecular weight is from about200 to about 500, from about 400 to about 5,000, from about 500 to about3,000, or from about 1,200 to about 3,000. The molecular weight of thePEG modification is from about 100, 200, 400, 500, 600, 800, 1,000,1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000,4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, to about15,000. Some non-limiting examples of lipids that may be used in thepresent application are taught by U.S. Patent 5,820,873, WO 2010/141069,or U.S. Patent 8,450,298, which is incorporated herein by reference.

In some embodiments of the lipid composition of the present application,the PEG lipid has a structural formula:

wherein: R₁₂ and R₁₃ are each independently alkyl_((C≤24)),alkenyl_((C≤24)), or a substituted version of either of these groups;R_(e) is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8)); and x is1-250. In some embodiments, R_(e) is alkyl_((C≤8)) such as methyl. R₁₂and R₁₃ are each independently alkyl_((C≤4-20)). In some embodiments, xis 5-250. In one embodiment, x is 5-125 or x is 100-250. In someembodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol,methoxypolyethylene glycol.

In some embodiments of the lipid composition of the present application,the PEG lipid has a structural formula:

wherein: n₁ is an integer between 1 and 100 and n₂ and n₃ are eachindependently selected from an integer between 1 and 29. In someembodiments, n₁ is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100, or any range derivable therein. In someembodiments, n₁ is from about 30 to about 50. In some embodiments, n₂ isfrom 5 to 23. In some embodiments, n₂ is 11 to about 17. In someembodiments, n₃ is from 5 to 23. In some embodiments, n₃ is 11 to about17.

In some embodiments of the lipid composition of the present application,the compositions may further comprise a molar percentage of the PEGlipid to the total lipid composition from about 4.0 to about 4.6. Insome embodiments, the molar percentage is from about 4.0, 4.1, 4.2, 4.3,4.4, 4.5, to about 4.6 or any range derivable therein. In otherembodiments, the molar percentage is from about 1.5 to about 4.0. Insome embodiments, the molar percentage is from about 1.5, 1.75, 2, 2.25,2.5, 2.75, 3, 3.25, 3.5, 3.75, to about 4.0 or any range derivabletherein.

In some embodiments of the lipid composition of the present application,the lipid composition comprises the polymer-conjugated lipid at a molarpercentage from about 0.5% to about 10%. In some embodiments of thelipid composition of the present application, the lipid compositioncomprises the polymer-conjugated lipid at a molar percentage from about1% to about 10%. In some embodiments of the lipid composition of thepresent application, the lipid composition comprises thepolymer-conjugated lipid at a molar percentage from about 2% to about10%. In some embodiments of the lipid composition of the presentapplication, the lipid composition comprises the polymer-conjugatedlipid at a molar percentage from about 1% to about 8%. In someembodiments of the lipid composition of the present application, thelipid composition comprises the polymer-conjugated lipid at a molarpercentage from about 2% to about 7%. In some embodiments of the lipidcomposition of the present application, the lipid composition comprisesthe polymer-conjugated lipid at a molar percentage from about 3% toabout 5%. In some embodiments of the lipid composition of the presentapplication, the lipid composition comprises the polymer-conjugatedlipid at a molar percentage from about 5% to about 10%. In someembodiments of the lipid composition of the present application, thelipid composition comprises the polymer-conjugated lipid at a molarpercentage of at least (about) 0.5%, at least (about) 1%, at least(about) 1.5%, at least (about) 2%, at least (about) 2.5%, at least(about) 3%, at least (about) 3.5%, at least (about) 4%, at least (about)4.5%, at least (about) 5%, at least (about) 5.5%, at least (about) 6%,at least (about) 6.5%, at least (about) 7%, at least (about) 7.5%, atleast (about) 8%, at least (about) 8.5%, at least (about) 9%, at least(about) 9.5%, or at least (about) 10%. In some embodiments of the lipidcomposition of the present application, the lipid composition comprisesthe polymer-conjugated lipid at a molar percentage of at most (about)0.5%, at most (about) 1%, at most (about) 1.5%, at most (about) 2%, atmost (about) 2.5%, at most (about) 3%, at most (about) 3.5%, at most(about) 4%, at most (about) 4.5%, at most (about) 5%, at most (about)5.5%, at most (about) 6%, at most (about) 6.5%, at most (about) 7%, atmost (about) 7.5%, at most (about) 8%, at most (about) 8.5%, at most(about) 9%, at most (about) 9.5%, or at most (about) 10%.

PHARMACEUTICAL COMPOSITIONS Therapeutic or Prophylactic Agents

In another aspect, provided herein is a pharmaceutical compositioncomprising a therapeutic agent (or prophylactic agent) assembled with alipid composition as described herein.

In some embodiments of the pharmaceutical composition, the therapeuticagent (or prophylactic agent) comprises a compound, a polynucleotide, apolypeptide, or a combination thereof. In some embodiments, thecompound, the polynucleotide, the polypeptide, or a combination thereofis exogenous or heterologous to the cell or the subject being treated bythe pharmaceutical compositions described herein. In some embodiments,the therapeutic agent (or prophylactic agent) comprises a compounddescribed herein. In some embodiments, the therapeutic agent (orprophylactic agent) comprises a polynucleotide described herein. In someembodiments, the therapeutic agent (or prophylactic agent) comprises apolypeptide described herein. In some embodiments, the therapeutic agent(or prophylactic agent) comprises a compound, a polynucleotide, apolypeptide, or a combination thereof.

In some embodiments, the pharmaceutical composition comprises atherapeutic agent (or prophylactic agent) for treating a lung diseasesuch as asthma, COPD, or lung cancer. In some embodiments, thetherapeutic agent (or prophylactic agent) comprises a steroid such asprednisone, hydrocortisone, prednisolone, methylprednisolone, ordexamethasone. In some embodiments, the therapeutic agent (orprophylactic agent) comprises Abraxane, Afatinib Dimaleate, Afinitor,Afinitor Disperz, Alecensa, Alectinib, Alimta, Alunbrig, Atezolizumab,Avastin, Bevacizumab, Brigatinib, Capmatinib Hydrochloride, Carboplatin,Ceritinib, Crizotinib, Cyramza, Dabrafenib Mesylate, Dacomitinib,Docetaxel, Doxorubicin Hydrochloride, Durvalumab, Entrectinib, ErlotinibHydrochloride, Everolimus, Gavreto, Gefitinib, Gilotrif, Gemcitabine,Ipilimumab, Iressa, Keytruda, Lorbrena, Mekinist, Methotrexate Sodium,Necitumumab, Nivolumab, Osimertinib Mesylate, Paclitaxel, Pembrolizumab,Pemetrexed Disodium, Pralsetinib, Ramucirumab, Retevmo, Selpercatinib,Tabrecta, Tafinlar, Tagrisso, Trametinib Dimethyl Sulfoxide, Vizimpro,Vinorelbine Tartrate, Xalkori, Yervoy, Zirabev, Zykadia, Carboplatin,Gemcitabine-cisplatin, Afinitor, Atezolizumab, Durvalumab, Etopophos,Etoposide, Hycamtin, Imfinzi, Keytruda, Lurbinectedin, MethotrexateSodium, Nivolumab, Opdivo, Pembrolizumab, Tecentriq, TopotecanHydrochloride, Trexall, or Zepzelca. Other non-limiting examples of thetherapeutic agents (or prophylactic agents) comprising compounds includesmall molecule selected from 7-Methoxypteridine, 7 Methylpteridine,abacavir, abafungin, abarelix, acebutolol, acenaphthene, acetaminophen,acetanilide, acetazolamide, acetohexamide, acetretin, acrivastine,adenine, adenosine, alatrofloxacin, albendazole, albuterol, alclofenac,aldesleukin, alemtuzumab, alfuzosin, alitretinoin, allobarbital,allopurinol, all-transretinoic acid (ATRA), aloxiprin, alprazolam,alprenolol, altretamine, amifostine, amiloride, aminoglutethimide,aminopyrine, amiodarone HCl, amitriptyline, amlodipine, amobarbital,amodiaquine, amoxapine, amphetamine, amphotericin, amphotericin B,ampicillin, amprenavir, amsacrine, amylnitrate, amylobarbitone,anastrozole, anrinone, anthracene, anthracyclines, aprobarbital, arsenictrioxide, asparaginase, aspirin, astemizole, atenolol, atorvastatin,atovaquone, atrazine, atropine, atropine azathioprine, auranofin,azacitidine, azapropazone, azathioprine, azintamide, azithromycin,aztreonum, baclofen, barbitone, BCG live, beclamide, beclomethasone,bendroflumethiazide, benezepril, benidipine, benorylate, benperidol,bentazepam, benzamide, benzanthracene, benzathine penicillin, benzhexolHCl, benznidazole, benzodiazepines, benzoic acid, bepheniumhydroxynaphthoate, betamethasone, bevacizumab (avastin), bexarotene,bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bisantrene,bleomycin, bleomycin, bortezomib, brinzolamide, bromazepam,bromocriptine mesylate, bromperidol, brotizolam, budesonide, bumetanide,bupropion, busulfan, butalbital, butamben, butenafine HCl,butobarbitone, butobarbitone (butethal), butoconazole, butoconazolenitrate, butylparaben, caffeine, calcifediol, calciprotriene,calcitriol, calusterone, cambendazole, camphor, camptothecin,camptothecin analogs, candesartan, capecitabine, capsaicin, captopril,carbamazepine, carbimazole, carbofuran, carboplatin, carbromal,carimazole, carmustine, cefamandole, cefazolin, cefixime, ceftazidime,cefuroxime axetil, celecoxib, cephradine, cerivastatin, cetrizine,cetuximab, chlorambucil, chloramphenicol, chlordiazepoxide,chlormethiazole, chloroquine, chlorothiazide, chlorpheniramine,chlorproguanil HCl, chlorpromazine, chlorpropamide, chlorprothixene,chlorpyrifos, chlortetracycline, chlorthalidone, chlorzoxazone,cholecalciferol, chrysene, cilostazol, cimetidine, cinnarizine,cinoxacin, ciprofibrate, ciprofloxacin HCl, cisapride, cisplatin,citalopram, cladribine, clarithromycin, clemastine fumarate, clioquinol,clobazam, clofarabine, clofazimine, clofibrate, clomiphene citrate,clomipramine, clonazepam, clopidogrel, clotiazepam, clotrimazole,clotrimazole, cloxacillin, clozapine, cocaine, codeine, colchicine,colistin, conjugated estrogens, corticosterone, cortisone, cortisoneacetate, cyclizine, cyclobarbital, cyclobenzaprine,cyclobutane-spirobarbiturate, cycloethane-spirobarbiturate,cycloheptane-spirobarbiturate, cyclohexane-spirobarbiturate,cyclopentane-spirobarbiturate, cyclophosphamide,cyclopropane-spirobarbiturate, cycloserine, cyclosporin, cyproheptadine,cytarabine, cytosine, dacarbazine, dactinomycin, danazol, danthron,dantrolene sodium, dapsone, darbepoetin alfa, darodipine, daunorubicin,decoquinate, dehydroepiandrosterone, delavirdine, demeclocycline,denileukin, deoxycorticosterone, desoxymethasone, dexamethasone,dexamphetamine, dexchlorpheniramine, dexfenfluramine, dexrazoxane,dextropropoxyphene, diamorphine, diatrizoicacid, diazepam, diazoxide,dichlorophen, dichlorprop, diclofenac, dicumarol, didanosine,diflunisal, digitoxin, digoxin, dihydrocodeine, dihydroequilin,dihydroergotamine mesylate, diiodohydroxyquinoline, diltiazem HCl,diloxamide furoate, dimenhydrinate, dimorpholamine, dinitolmide,diosgenin, diphenoxylate HCl, diphenyl, dipyridamole, dirithromycin,disopyramide, disulfiram, diuron, docetaxel, domperidone, donepezil,doxazosin, doxazosin HCl, doxorubicin, doxycycline, dromostanolonepropionate, droperidol, dyphylline, echinocandins, econazole, econazolenitrate, efavirenz, ellipticine, enalapril, enlimomab, enoximone,epinephrine, epipodophyllotoxin derivatives, epirubicin, epoetinalfa,eposartan, equilenin, equilin, ergocalciferol, ergotamine tartrate,erlotinib, erythromycin, estradiol, estramustine, estriol, estrone,ethacrynic acid, ethambutol, ethinamate, ethionamide, ethopropazine HCl,ethyl-4-aminobenzoate (benzocaine), ethylparaben, ethinylestradiol,etodolac, etomidate, etoposide, etretinate, exemestane, felbamate,felodipine, fenbendazole, fenbuconazole, fenbufen, fenchlorphos,fenclofenac, fenfluramine, fenofibrate, fenoldepam, fenoprofen calcium,fenoxycarb, fenpiclonil, fentanyl, fenticonazole, fexofenadine,filgrastim, finasteride, flecamide acetate, floxuridine, fludarabine,fluconazole, fluconazole, flucytosine, fludioxonil, fludrocortisone,fludrocortisone acetate, flufenamic acid, flunanisone, flunarizine HCl,flunisolide, flunitrazepam, fluocortolone, fluometuron, fluorene,fluorouracil, fluoxetine HCl, fluoxymesterone, flupenthixol decanoate,fluphenthixol decanoate, flurazepam, flurbiprofen, fluticasonepropionate, fluvastatin, folic acid, fosenopril, fosphenytoin sodium,frovatriptan, furosemide, fulvestrant, furazolidone, gabapentin, G-BHC(Lindane), gefitinib, gemcitabine, gemfibrozil, gemtuzumab, glafenine,glibenclamide, gliclazide, glimepiride, glipizide, glutethimide,glyburide, Glyceryltrinitrate (nitroglycerin), goserelin acetate,grepafloxacin, griseofulvin, guaifenesin, guanabenz acetate, guanine,halofantrine HCl, haloperidol, hydrochlorothiazide, heptabarbital,heroin, hesperetin, hexachlorobenzene, hexethal, histrelin acetate,hydrocortisone, hydroflumethiazide, hydroxyurea, hyoscyamine,hypoxanthine, ibritumomab, ibuprofen, idarubicin, idobutal, ifosfamide,ihydroequilenin, imatinib mesylate, imipenem, indapamide, indinavir,indomethacin, indoprofen, interferon alfa-2a, interferon alfa-2b,iodamide, iopanoic acid, iprodione, irbesartan, irinotecan,isavuconazole, isocarboxazid, isoconazole, isoguanine, isoniazid,isopropylbarbiturate, isoproturon, isosorbide dinitrate, isosorbidemononitrate, isradipine, itraconazole, itraconazole, itraconazole(Itra), ivermectin, ketoconazole, ketoprofen, ketorolac, khellin,labetalol, lamivudine, lamotrigine, lanatoside C, lanosprazole, L-DOPA,leflunomide, lenalidomide, letrozole, leucovorin, leuprolide acetate,levamisole, levofloxacin, lidocaine, linuron, lisinopril, lomefloxacin,lomustine, loperamide, loratadine, lorazepam, lorefloxacin,lormetazepam, losartan mesylate, lovastatin, lysuride maleate,Maprotiline HCl, mazindol, mebendazole, Meclizine HCl, meclofenamicacid, medazepam, medigoxin, medroxyprogesterone acetate, mefenamic acid,Mefloquine HCl, megestrol acetate, melphalan, mepenzolate bromide,meprobamate, meptazinol, mercaptopurine, mesalazine, mesna,mesoridazine, mestranol, methadone, methaqualone, methocarbamol,methoin, methotrexate, methoxsalen, methsuximide, methyclothiazide,methylphenidate, methylphenobarbitone, methyl-p-hydroxybenzoate,methylprednisolone, methyltestosterone, methyprylon, methysergidemaleate, metoclopramide, metolazone, metoprolol, metronidazole,Mianserin HCl, miconazole, midazolam, mifepristone, miglitol,minocycline, minoxidil, mitomycin C, mitotane, mitoxantrone,mofetilmycophenolate, molindone, montelukast, morphine, MoxifloxacinHCl, nabumetone, nadolol, nalbuphine, nalidixic acid, nandrolone,naphthacene, naphthalene, naproxen, naratriptan HCl, natamycin,nelarabine, nelfinavir, nevirapine, nicardipine HCl, nicotin amide,nicotinic acid, nicoumalone, nifedipine, nilutamide, nimodipine,nimorazole, nisoldipine, nitrazepam, nitrofurantoin, nitrofurazone,nizatidine, nofetumomab, norethisterone, norfloxacin, norgestrel,nortriptyline HCl, nystatin, oestradiol, ofloxacin, olanzapine,omeprazole, omoconazole, ondansetron HCl, oprelvekin, ornidazole,oxaliplatin, oxamniquine, oxantelembonate, oxaprozin, oxatomide,oxazepam, oxcarbazepine, oxfendazole, oxiconazole, oxprenolol,oxyphenbutazone, oxyphencyclimine HCl, paclitaxel, palifermin,pamidronate, p-aminosalicylic acid, pantoprazole, paramethadione,paroxetine HCl, pegademase, pegaspargase, pegfilgrastim,pemetrexeddisodium, penicillamine, pentaerythritol tetranitrate,pentazocin, pentazocine, pentobarbital, pentobarbitone, pentostatin,pentoxifylline, perphenazine, perphenazine pimozide, perylene,phenacemide, phenacetin, phenanthrene, phenindione, phenobarbital,phenolbarbitone, phenolphthalein, phenoxybenzamine, phenoxybenzamineHCl, phenoxymethyl penicillin, phensuximide, phenylbutazone, phenytoin,pindolol, pioglitazone, pipobroman, piroxicam, pizotifen maleate,platinum compounds, plicamycin, polyenes, polymyxin B, porfimersodium,posaconazole (Posa), pramipexole, prasterone, pravastatin, praziquantel,prazosin, prazosin HCl, prednisolone, prednisone, primidone,probarbital, probenecid, probucol, procarbazine, prochlorperazine,progesterone, proguanil HCl, promethazine, propofol, propoxur,propranolol, propylparaben, propylthiouracil, prostaglandin,pseudoephedrine, pteridine-2-methyl-thiol, pteridine-2-thiol,pteridine-4-methyl-thiol, pteridine-4-thiol, pteridine-7-methyl-thiol,pteridine-7-thiol, pyrantelembonate, pyrazinamide, pyrene,pyridostigmine, pyrimethamine, quetiapine, quinacrine, quinapril,quinidine, quinidine sulfate, quinine, quininesulfate, rabeprazolesodium, ranitidine HCl, rasburicase, ravuconazole, repaglinide, reposal,reserpine, retinoids, rifabutine, rifampicin, rifapentine, rimexolone,risperidone, ritonavir, rituximab, rizatriptan benzoate, rofecoxib,ropinirole HCl, rosiglitazone, saccharin, salbutamol, salicylamide,salicylic acid, saquinavir, sargramostim, secbutabarbital, secobarbital,sertaconazole, sertindole, sertraline HCl, simvastatin, sirolimus,sorafenib, sparfloxacin, spiramycin, spironolactone, stanolone,stanozolol, stavudine, stilbestrol, streptozocin, strychnine,sulconazole, sulconazole nitrate, sulfacetamide, sulfadiazine,sulfamerazine, sulfamethazine, sulfamethoxazole, sulfanilamide,sulfathiazole, sulindac, sulphabenzamide, sulphacetamide, sulphadiazine,sulphadoxine, sulphafurazole, sulphamerazine, sulpha-methoxazole,sulphapyridine, sulphasalazine, sulphinpyrazone, sulpiride, sulthiame,sumatriptan succinate, sunitinib maleate, tacrine, tacrolimus, talbutal,tamoxifen citrate, tamulosin, targretin, taxanes, tazarotene,telmisartan, temazepam, temozolomide, teniposide, tenoxicam, terazosin,terazosin HCl, terbinafine HCl, terbutaline sulfate, terconazole,terfenadine, testolactone, testosterone, tetracycline,tetrahydrocannabinol, tetroxoprim, thalidomide, thebaine, theobromine,theophylline, thiabendazole, thiamphenicol, thioguanine, thioridazine,thiotepa, thotoin, thymine, tiagabine HCl, tibolone, ticlopidine,tinidazole, tioconazole, tirofiban, tizanidine HCl, tolazamide,tolbutamide, tolcapone, topiramate, topotecan, toremifene, tositumomab,tramadol, trastuzumab, trazodone HCl, tretinoin, triamcinolone,triamterene, triazolam, triazoles, triflupromazine, trimethoprim,trimipramine maleate, triphenylene, troglitazone, tromethamine,tropicamide, trovafloxacin, tybamate, ubidecarenone (coenzyme Q10),undecenoic acid, uracil, uracil mustard, uric acid, valproic acid,valrubicin, valsartan, vancomycin, venlafaxine HCl, vigabatrin,vinbarbital, vinblastine, vincristine, vinorelbine, voriconazole,xanthine, zafirlukast, zidovudine, zileuton, zoledronate, zoledronicacid, zolmitriptan, zolpidem, or zopiclone.

Polynucleotides

In some embodiments of the pharmaceutical compositions of the presentapplication, the therapeutic agent (or prophylactic agent) assembledwith the lipid composition comprises one or more polynucleotides. Thepresent application is not limited in scope to any particular source,sequence, or type of polynucleotide; however, as one of ordinary skillin the art could readily identify related homologs in various othersources of the polynucleotide including nucleic acids from non-humanspecies (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimp, ape,baboon, cow, pig, horse, sheep, cat and other species). It iscontemplated that the polynucleotide used in the present application cancomprises a sequence based upon a naturally-occurring sequence. Allowingfor the degeneracy of the genetic code, sequences that have at leastabout 50%, usually at least about 60%, more usually about 70%, mostusually about 80%, preferably at least about 90% and most preferablyabout 95% of nucleotides that are identical to the nucleotide sequenceof the naturally-occurring sequence. In another embodiment, thepolynucleotide comprises nucleic acid sequence that is a complementarysequence to a naturally occurring sequence, or complementary to 75%,80%, 85%, 90%, 95% and 100%. Longer polynucleotides encoding 250, 500,1000, 1212, 1500, 2000, 2500, 3000 or longer are contemplated herein.

In some embodiments, the polynucleotide used herein may be derived fromgenomic DNA, i.e., cloned directly from the genome of a particularorganism. In preferred embodiments, however, the polynucleotide wouldcomprise complementary DNA (cDNA). Also contemplated is a cDNA plus anatural intron or an intron derived from another gene; such engineeredmolecules are sometime referred to as “mini-genes.” At a minimum, theseand other nucleic acids of the present application may be used asmolecular weight standards in, for example, gel electrophoresis. Theterm “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

In some embodiments, the polynucleotide comprises one or more segmentscomprising a small interfering ribonucleic acid (siRNA), a short hairpinRNA (shRNA), a micro-ribonucleic acid (miRNA), a primarymicro-ribonucleic acid (pri-miRNA), a long non-coding RNA (IncRNA), amessenger ribonucleic acid (mRNA), a clustered regularly interspacedshort palindromic repeats (CRISPR) related nucleic acid, a CRISPR-RNA(crRNA), a single guide ribonucleic acid (sgRNA), a trans-activatingCRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid(pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide(ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid,deoxyribonucleic acid (DNA), a double stranded deoxyribonucleic acid(dsDNA), a single stranded deoxyribonucleic acid (ssDNA), a singlestranded ribonucleic acid (ssRNA), a or double stranded ribonucleic acid(dsRNA). In some embodiments, the polynucleotide encodes at least one ofthe therapeutic agent (or prophylactic agent) described herein. In someembodiments, the polynucleotide encodes at least one guidepolynucleotide, such as guide RNA (gRNA) or guide DNA (gDNA), forcomplexing with a guide RNA guided nuclease described herein. In someembodiments, the polynucleotide encodes at least one guidepolynucleotide guided heterologous nuclease. The nuclease may be anendonuclease. Non-limiting example of the guide polynucleotide guidedheterologous endonuclease may be selected from CRISPR-associated (Cas)proteins or Cas nucleases including type I CRISPR-associated (Cas)polypeptides, type II CRISPR-associated (Cas) polypeptides, type IIICRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas)polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VICRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN);transcription activator-like effector nucleases (TALEN); meganucleases;RNA-binding proteins (RBP); CRISPR-associated RNA binding proteins;recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g.,prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryoticArgonaute (eAgo), and Natronobacterium gregoryi Argonaute (NgAgo));Adenosine deaminases acting on RNA (ADAR); CIRT, PUF, homingendonuclease, or any functional fragment thereof, any derivativethereof; any variant thereof; and any fragment thereof.

In some embodiments, the therapeutic (or prophylactic) agent is atransfer ribonucleic acid (tRNA) that introduces an amino acid into agrowing peptide chain of a protein of a target gene. The target gene canbe one set forth in Table 7.

TABLE 7 Example target genes for transfer RNA therapy Target Gene NameCorresponding Protein Disease CFTR CFTR Cystic fibrosis transmembraneconductance regulator Cystic fibrosis DNAH5 DNAH5 Dynein axonemal heavychain 5 Primary ciliary dyskinesia DNAH11 DNAH11 Dynein axonemal heavychain 11 Primary ciliary dyskinesia BMPR2 BMPR2 Bone morphogeneticprotein receptor type 2 Pulmonary arterial hypertension FAH FAHFumarylacetoacetate hydrolase Tyrosinemia PAH PAH Phenylalaninehydroxylase Phenylketonuria IDUA IDUA Alpha-L-iduronidaseMucopolysaccharidosis i COL4A3 COL4A3 Collagen type IV alpha 3 chainAlport syndrome COL4A4 COL4A4 Collagen type IV alpha 4 chain Alportsyndrome COL4A5 COL4A5 Collagen type IV alpha 5 chain Alport syndromePKD1 PKD1 Polycystin 1 Polycystic kidney disease PKD2 PKD2 Polycystin 2Polycystic kidney disease PKHD1 PKHD1 Fibrocystin (or polyductin)Polycystic kidney disease SLC3A1 SLC3A1 Solute carrier family 3 member 1Cystinuria SLC7A9 SLC7A9 Solute carrier family 7 member 9 CystinuriaPAX9 PAX9 Paired box gene 9 Aniridia MYO7A MYO7A Myosin VIIA Ushersyndrome CDH23 CDH23 Cadherin related 23 Usher syndrome USH2A USH2AUsherin Usher syndrome CLRN1 CLRN1 Clarin 1 Usher syndrome GJB2 GJB2 Gapjunction beta-2 protein Non-syndromic hearing loss GJB6 GJB6 Gapjunction beta-6 protein Non-syndromic hearing loss RHO RHO RhodopsinRetinitis pigmentosa DMPK DMPK dystrophia myotonica protein kinaseMyotonic dystrophy type 1 DMD DMD Dystrophin Muscular dystrophy SCN1ASCN1A Sodium voltage-gated channel alpha subunit 1 Dravet syndrome SCN1BSCN1B Sodium voltage-gated channel beta subunit 1 Dravet syndrome F8 F8Coagulation factor VIII Hemophilia a F9 F9 Coagulation factor IXHemophilia b NGLY1 NGLY1 N-glycanase 1 N-glycanase 1 deficiency p53 p53Tumor protein p53 Cancer CLN1 PPT1 Palmitoyl-protein thioesterase 1Batten disease CLN2 TPP1 Tripeptidyl peptidase 1 Late infantile ceroidlipofuscinoses HERG hERG Kv11.1 (alpha subunit of potassium ion channel)Long qt syndrome PPT1 PPT1 Palmitoyl-protein thioesterase 1 Infantileceroid lipofuscinoses ATM ATM ATM serine/threonine kinase Ataxiatelangiectasia FBN1 FBN1 Fibrillin 1 Usher syndrome type 1

Some embodiments of the therapeutic agent (or prophylactic agent)provided herein comprise a heterologous polypeptide comprising anactuator moiety. The actuator moiety can be configured to complex with atarget polynucleotide corresponding to a target gene. In someembodiments, administration of the therapeutic agent (or prophylacticagent) results in a modified expression or activity of the target gene.The modified expression or activity of the target gene can bedetectable, for example, in at least about 5%, 10%, 15%, or 20% lungepithelial cells of said subject, in at least about 2%, 5%, or 10% lungciliated cells of said subject, in at least about 5%, 10%, 15%, or 20%lung secretory cells of said subject, in at least about 5%, 10%, 15%, or20% lung club cells of said subject, in at least about 5%, 10%, 15%, or20% lung goblet cells of said subject, or in at least about 5%, 10%,15%, or 20% lung basal cells of said subject, or a combination thereof.Biomarkers for characterizing the cell types might be known in thefield, such as those described herein below in the Examples section. Thetherapeutic agent (or prophylactic agent) may comprise a heterologouspolynucleotide encoding an actuator moiety. The actuator moiety may beconfigured to complex with a target polynucleotide corresponding to atarget gene. The heterologous polynucleotide may encode a guidepolynucleotide configured to direct the actuator moiety to the targetpolynucleotide. The actuator moiety may comprise a heterologousendonuclease or a fragment thereof (e.g., directed by a guidepolynucleotide to specifically bind the target polynucleotide). Theheterologous endonuclease may be (1) part of a ribonucleoprotein (RNP)and (2) complexed with the guide polynucleotide. The heterologousendonuclease may be part of a clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) protein complex.The heterologous endonuclease may be a clustered regularly interspacedshort palindromic repeats (CRISPR)-associated (Cas) endonuclease. Theheterologous endonuclease may comprise a deactivated endonuclease. Thedeactivated endonuclease may be fused to a regulatory moiety. Theregulatory moiety may comprise a transcription activator, atranscription repressor, an epigenetic modifier, or a fragment thereof.

In some embodiments, the polynucleotide encodes at least one guidepolynucleotide (such as guide RNA (gRNA) or guide DNA (gDNA)) guidedheterologous endonuclease. In some embodiments, the polynucleotideencodes at least one guide polynucleotide and at least one heterologousendonuclease, where the guide polynucleotide can be complexed with andguides the at least one heterologous endonuclease to cleave a geneticlocus of any one of the genes described herein. In some embodiments, thepolynucleotide encodes at least one guide polynucleotide guidedheterologous endonuclease such as Cas9, Cas12, Cas13, Cpf1 (or Cas12a),C2C1, C2C2 (or Cas13a), Cas13b, Cas13c, Cas13d, Cas14, C2C3, Casl,CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7,Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Csnl, Csx12, Cas10, Cas10d, CaslO,CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB),Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4,Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7,Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, orCul966; any derivative thereof; any variant thereof; or any fragmentthereof. In some embodiments, Cas13 can include, but are not limited to,Cas13a, Cas13b, Cas13c, and Cas 13d (e.g., CasRx).

In some embodiments, the heterologous endonuclease comprises adeactivated endonuclease, optionally fused to a regulatory moiety, suchas an epigenetic modifier to remodel the epigenome that mediates theexpression of the selected genes of interest. In some cases, theepigenetic modifier can include methyltransferase, demethylase,dismutase, an alkylating enzyme, depurinase, oxidase, photolyase,integrase, transposase, recombinase, polymerase, ligase, helicase,glycosylase, acetyltransferase, deacetylase, kinase, phosphatase,ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, ubiquitinligase, deubiquitinating enzyme, adenylate-forming enzyme, AMPylator,de-AMPylator, SUMOylating enzyme, deSUMOylating enzyme, ribosylase,deribosylase, N-myristoyltransferase, chromotine remodeling enzyme,protease, oxidoreductase, transferase, hydrolase, lyase, isomerase,synthase, synthetase, or demyristoylation enzyme. In some instances, theepigenetic modifier can comprise one or more selected from the groupconsisting of p300, TET1, LSD1, HDAC1, HDAC8, HDAC4, HDAC11, HDT1,SIRT3, HST2, CobB, SIRT5, SIR2A, SIRT6, NUE, vSET, SUV39H1, DIM5, KYP,SUVR4, Set4, Set1, SETD8, and TgSET8.

In some embodiments, the polynucleotide encodes a guide polynucleotide(such as guide RNA (gRNA) or guide DNA (gDNA)) that is at leastpartially complementary to the genomic region of a gene, where uponbinding of the guide polynucleotide to the gene the guide polynucleotiderecruits the guide polynucleotide guided nuclease to cleave andgenetically modified the region. Examples of the genes that may bemodified by the guide polynucleotide guided nuclease include CFTR,DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1,PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2,GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1,hERG, PPT1, ATM, or FBN1.

In some embodiments, the polynucleotide comprises or encodes at leastone mRNA that, upon expression of the mRNA, restores the function of adefective gene in a subject being treated by the pharmaceuticalcomposition described herein. For example, the polynucleotide comprisesor encodes an mRNA that expresses a wild type CFTR protein, which may beused to rescue a subject who is afflicted with inborn mutation in CFTRprotein. Other examples of mRNA that can be expressed from thepolynucleotide includes mRNA that encodes DNAH5, DNAH11, BMPR2, FAH,PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9,PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A,SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.

In some embodiments, the polynucleotides of the present applicationcomprise at least one chemical modifications of the one or morenucleotides. In some embodiments, the chemical modification increasesspecificity of the guide polynucleotide (such as guide RNA (gRNA) orguide DNA (gDNA)) binding to a complementary genomic locus (e.g., thegenomic locus of any one of the genes described herein). In someembodiments, the at least one chemical modification increases resistanceto nuclease digestion, when then polynucleotide is administered to asubject in need thereof. In some embodiments, the at least one chemicalmodification decreases immunogenicity, when then polynucleotide isadministered to a subject in need thereof. In some embodiments, the atleast one chemical modification stabilizes scaffold such as a tRNAscaffold. Such chemical modification may have desirable properties, suchas enhanced resistance to nuclease digestion or increased bindingaffinity with a target genomic locus relative to a polynucleotidewithout the at least one chemical modification.

In some embodiments, the at least one chemical modification comprisesmodification to sugar moiety. In some embodiments, modified sugarmoieties are substituted sugar moieties comprising one or morenon-bridging sugar substituent, including but not limited tosubstituents at the 2′ and/or 5′ positions. Examples of sugarsubstituents suitable for the 2′-position, include, but are not limitedto: 2′—F, 2′—OCH₃ (“OMe” or “O-methyl”), and 2′—O(CH₂)₂OCH₃ (“MOE”). Incertain embodiments, sugar substituents at the 2′ position is selectedfrom allyl, amino, azido, thio, O-allyl, O--C₁-C₁₀ alkyl, O--C₁-C₁₀substituted alkyl; OCF₃, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), andO—CH₂—C(═O)—N(R_(m))(R_(n)), where each Rm and Rn is, independently, Hor substituted or unsubstituted C₁-C₁₀ alkyl. Examples of sugarsubstituents at the 5′-position, include, but are not limited to:5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In some embodiments,substituted sugars comprise more than one non-bridging sugarsubstituent, for example, T-F-5′-methyl sugar moieties.

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In some embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(R_(m))-alkyl; O, S, orN(R_(m))-alkenyl; O, S or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl,alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H, an amino protecting group orsubstituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituent groupscan be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In some embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In some embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, andO—CH₂—C(═O)—N(H)CH₃.

In some embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. In somesuch embodiments, the bicyclic sugar moiety comprises a bridge betweenthe 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ sugarsubstituents, include, but are not limited to: —[C(R_(a))(R_(b))]_(n)—,—[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O— or,—C(R_(a)R_(b))—O—N(R)--; 4′—CH₂—2′, 4′—(CH₂)₂—2′, 4′—(CH₂)—O—2′ (LNA);4′—(CH₂)—S—2′; 4′—(CH₂)₂—O—2′ (ENA); 4′—CH(CH₃)—O—2′ (cEt) and4′—CH(CH₂OCH₃)—O—2′, and analogs thereof; 4′—C(CH₃)(CH₃)—O—2′ andanalogs thereof; 4′-CH₂—N(OCH₃)—2′ and analogs thereof;4′—CH₂—O—N(CH₃)—2′; 4′—CH₂—O—N(R)-2′, and 4′—CH₂—N(R)—O—2′—, whereineach R is, independently, H, a protecting group, or C₁-C₁₂ alkyl;4′—CH₂—N(R)—O—2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group;4′—CH₂—C(H)(CH₃)—2′; and 4′—CH₂—C(═CH₂)—2′ and analogs thereof.

In some embodiments, such 4′ to 2′ bridges independently comprise from 1to 4 linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—; wherein: x is 0, 1, or 2; nis 1, 2, 3, or 4; each R_(a) and R_(b) is, independently, H, aprotecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycleradical, substituted heterocycle radical, heteroaryl, substitutedheteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclicradical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H),substituted acyl, CN, sulfonyl (S(═O)₂—J₁), or sulfoxyl (S(═O)—J₁); andeach J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α—L—Methyleneoxy (4′—CH₂—O—2′) BNA, (B) β—D—Methyleneoxy(4′—CH₂—O—2′) BNA (also referred to as locked nucleic acid or LNA), (C)Ethyleneoxy (4′—(CH₂)₂—O—2′) BNA, (D) Aminooxy (4′—CH₂—O—N(R)-2′) BNA,(E) Oxyamino (4′—CH₂—N(R)--O-2′) BNA, (F) Methyl(methyleneoxy)(4′—CH(CH₃)—O—2′) BNA (also referred to as constrained ethyl or cEt),(G) methylene-thio (4′—CH₂—S—2′) BNA, (H) methylene-amino(4′—CH2—N(R)-2′) BNA, (I) methyl carbocyclic (4′—CH₂—CH(CH₃)—2′) BNA,(J) propylene carbocyclic (4′—(CH₂)₃—2′) BNA, and (K)Methoxy(ethyleneoxy) (4′—CH(CH₂OMe)—O—2′) BNA (also referred to asconstrained MOE or cMOE).

In some embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the .alpha.-L configuration or in the.beta.-D configuration. Previously, α-L-methyleneoxy (4′—CH₂—O—2′)bicyclic nucleosides have been incorporated into antisensepolynucleotides that showed antisense activity.

In some embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars, wherein LNAis substituted with, for example, a 5′-methyl or a 5′-vinyl group).

In some embodiments, modified sugar moieties are sugar surrogates. Insome such embodiments, the oxygen atom of the naturally occurring sugaris substituted, e.g., with a sulfur, carbon or nitrogen atom. In somesuch embodiments, such modified sugar moiety also comprises bridgingand/or non-bridging substituents as described above. For example,certain sugar surrogates comprise a 4′-sulfur atom and a substitution atthe 2′-position and/or the 5′ position. By way of additional example,carbocyclic bicyclic nucleosides having a 4′-2′ bridge have beendescribed.

In some embodiments, sugar surrogates comprise rings having other than5-atoms. For example, in some embodiments, a sugar surrogate comprises asix-membered tetrahydropyran. Such tetrahydropyrans may be furthermodified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA), and fluoroHNA (F-HNA).

In some embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In some embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ ismethyl. In some embodiments, THP nucleosides of Formula VII are providedwherein one of R₁ and R₂ is F. In certain embodiments, R₁ is fluoro andR₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds.

Combinations of modifications are also provided without limitation, suchas 2′—F—5′-methyl substituted nucleosides and replacement of the ribosylring oxygen atom with S and further substitution at the 2′-position oralternatively 5′-substitution of a bicyclic nucleic acid. In someembodiments, a 4′—CH₂—O—2′ bicyclic nucleoside is further substituted atthe 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis andpreparation of carbocyclic bicyclic nucleosides along with theiroligomerization and biochemical studies have also been described.

In some embodiments, the present application provides polynucleotidecomprising modified nucleosides. Those modified nucleotides may includemodified sugars, modified nucleobases, and/or modified linkages. Thespecific modifications are selected such that the resultingpolynucleotide possesses desirable characteristics. In some embodiments,polynucleotide comprises one or more RNA-like nucleosides. In someembodiments, polynucleotide comprises one or more DNA-like nucleotides.

In some embodiments, nucleosides of the present application comprise oneor more unmodified nucleobases. In certain embodiments, nucleosides ofthe present application comprise one or more modified nucleobases.

In some embodiments, modified nucleobases are selected from: universalbases, hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynylCH₃) uracil and cytosine and other alkynyl derivatives of pyrimidinebases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine,3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases,promiscuous bases, size-expanded bases, and fluorinated bases as definedherein. Further modified nucleobases include tricyclic pyrimidines suchas phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one),carbazole cytidine (²H-pyrimido[4,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deazaadenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone.

In some embodiments, the present application provides poylnucleotidecomprising linked nucleosides. In such embodiments, nucleosides may belinked together using any internucleoside linkage. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus containinginternucleoside linkages include, but are not limited to,phosphodiesters (P=O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P=S). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Modified linkages, compared to natural phosphodiester linkages, can beused to alter, typically increase, nuclease resistance of theoligonucleotide. In some embodiments, internucleoside linkages having achiral atom can be prepared as a racemic mixture, or as separateenantiomers. Representative chiral linkages include, but are not limitedto, alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The polynucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), α or β such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′—CH₂—N(CH₃)—O—5′), amide-3(3′—CH₂—C(═O)—N(H)—5′), amide-4 (3′—CH₂—N(H)—C(═O)—5′), formacetal(3′—O—CH₂—O—5′), and thioformacetal (3′—S—CH₂—O—5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Forexample, one additional modification of the ligand conjugatedpolynucleotides of the present application involves chemically linkingto the oligonucleotide one or more additional non-ligand moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety, cholic acid, athioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,dihexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

In some embodiments, the polynucleotides described herein comprise orencode at least one tRNA described herein. In some embodiments, the tRNAexpressed from the polynucleotide restores the function of at least onedefective tRNA in a subject who is being treated by the pharmaceuticalcomposition described herein. In some embodiments, the at least one tRNAexpressed by the polynucleotide described herein may include tRNA thatencodes alanine, arginine, asparagine, aspartic acid, cysteine, glutamicacid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucin,lysine, methionine, phenylaniline, proline, pyroglutamic acid, serine,threonine, tryptophan, tyrosine, or valine. In some embodiments, the atleast one tRNA expressed by the polynucleotide described herein mayinclude tRNA that encodes arginine, tryptophan, glutamic acid,glutamine, serine, tyrosine, lysine, leucine, glycine, or cysteine. Insome embodiments, the tRNA encoded by the polynucleotide describedherein may restore the expression of any one of the genes describedherein. In some embodiments, the tRNA encoded by the polynucleotidedescribed herein may restore the expression of CFTR, DNAH5, DNAH11,BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1,SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK,DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, orFBN1. Polypeptides

In some embodiments of the pharmaceutical compositions of the presentapplication, the therapeutic agent (or prophylactic agent) assembledwith the lipid composition comprises one or more one or morepolypeptides. Some polypeptide may include enzymes such as any one ofthe nuclease enzymes described herein. For example, the nuclease enzymemay include from CRISPR-associated (Cas) proteins or Cas nucleasesincluding type I CRISPR-associated (Cas) polypeptides, type IICRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas)polypeptides, type IV CRISPR-associated (Cas) polypeptides, type VCRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated(Cas) polypeptides; zinc finger nucleases (ZFN); transcriptionactivator-like effector nucleases (TALEN); meganucleases; RNA-bindingproteins (RBP); CRISPR-associated RNA binding proteins; recombinases;flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryoticArgonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute(eAgo), and Natronobacterium gregoryi Argonaute (NgAgo)); Adenosinedeaminases acting on RNA (ADAR); CIRT, PUF, homing endonuclease, or anyfunctional fragment thereof, any derivative thereof; any variantthereof; and any fragment thereof. In some embodiments, the nucleaseenzyme may include Cas proteins such as Cas1, Cas1B, Cas2, Cas3, Cas4,Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10,Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4,Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4,homologs thereof, or modified versions thereof. In some embodiments, theCas protein may be complexed with a guide polynucleotide describedherein to be form a CRISPR ribonucleoprotein (RNP).

The nuclease in the compositions described herein may be Cas9 (e.g.,from S. pyogenes or S. pneumonia). The CRISPR enzyme can direct cleavageof one or both strands at the location of a target sequence, such aswithin the target sequence and/or within the complement of the targetsequence of any one of the genes described herein. For example, theCRISPR enzyme may be directed and cleaved a genomic locus of CFTR,DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1,PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2,GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1,hERG, PPT1, ATM, or FBN1.

The CRISPR enzyme may be mutated with respect to a correspondingwild-type enzyme such that the mutated CRISPR enzyme lacks the abilityto cleave one or both strands of a target polynucleotide containing atarget sequence. For example, an aspartate-to-alanine substitution(D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes convertsCas9 from a nuclease that cleaves both strands to a nickase (cleaves asingle strand). In some embodiments, a Cas9 nickase may be used incombination with guide sequence(s), e.g., two guide sequences, whichtarget respectively sense and antisense strands of the DNA target. Thiscombination allows both strands to be nicked and used to induce NHEJ orHDR.

In some embodiments, the present application provides polypeptidecontaining one or more therapeutic proteins. The therapeutic proteinsthat may be included in the composition include a wide range ofmolecules such as cytokines, chemokines, interleukins, interferons,growth factors, coagulation factors, anti-coagulants, blood factors,bone morphogenic proteins, immunoglobulins, and enzymes. Somenon-limiting examples of particular therapeutic proteins includeErythropoietin (EPO), Granulocyte colony-stimulating factor (G-CSF),Alpha-galactosidase A, Alpha-L-iduronidase, Thyrotropin α,N-acetylgalactosamine-4-sulfatase (rhASB), Dornase alfa, Tissueplasminogen activator (TPA) Activase, Glucocerebrosidase, Interferon(IF) β-1a, Interferon β-1b, Interferon γ, Interferon α, TNF-α, IL-1through IL-36, Human growth hormone (rHGH), Human insulin (BHI), Humanchorionic gonadotropin α, Darbepoetin α, Follicle-stimulating hormone(FSH), and Factor VIII.

In some embodiments, the polypeptide comprises a peptide sequence thatis at least partially identical to any of the therapeutic agent (orprophylactic agent) comprising a peptide sequence. For example, thepolypeptide may comprise a peptide sequence that is at least partiallyidentical to an antibody (e.g., a monoclonal antibody) for treating alung disease such as lung cancer.

In some embodiments, the polypeptide comprises a peptide or protein thatrestores the function of a defective protein in a subject being treatedby the pharmaceutical composition described herein. For example, thepolynucleotide comprises a peptide or protein that restores function ofcystic fibrosis transmembrane conductance regulator (CFTR) protein,which may be used to rescue a subject who is afflicted with inborn errorleading to the expression of the mutated CFTR protein. Other examples ofthe rescue may include administering to a subject in need thereof apolypeptide comprising a peptide or protein of wild type Dynein axonemalheavy chain 5, Dynein axonemal heavy chain 11,Bone morphogenetic proteinreceptor type 2,Fumarylacetoacetate hydrolase, Phenylalaninehydroxylase, Alpha-L-iduronidase,Collagen type IV alpha 3 chain,Collagen type IV alpha 4 chain, Collagen type IV alpha 5chain,Polycystin 1, Polycystin 2, Fibrocystin (or polyductin), Solutecarrier family 3 member 1,Solute carrier family 7 member 9,Paired boxgene 9,Myosin VIIA, Cadherin related 23, Usherin, Clarin 1, Gap junctionbeta-2 protein, Gap junction beta-6 protein, Rhodopsin, dystrophiamyotonica protein kinase, Dystrophin, Sodium voltage-gated channel alphasubunit 1, Sodium voltage-gated channel beta subunit 1, Coagulationfactor VIII, Coagulation factor IX, N-glycanase 1, Tumor protein p53,Palmitoyl-protein thioesterase 1, Tripeptidyl peptidase 1,Kv11.1 (alphasubunit of potassium ion channel), Palmitoyl-protein thioesterase 1, ATMserine/threonine kinase, or Fibrillin 1.

In some embodiments, the pharmaceutical composition of the presentapplication comprises a plurality of payloads assembled with (e.g.,encapsulated within) a lipid composition. The plurality of payloadsassembled with the lipid composition may be configured for gene-editingor gene-expression modification. The plurality of payloads assembledwith the lipid composition may comprise a polynucleotide encoding anactuator moiety (e.g., comprising a heterologous endonuclease such asCas) or a polynucleotide encoding the actuator moiety. The plurality ofpayloads assembled with the lipid composition may further comprise oneor more (e.g., one or two) guide polynucleotides. The plurality ofpayloads assembled with the lipid composition may further comprise oneor more donor or template polynucleotides. The plurality of payloadsassembled with the lipid composition may comprise a ribonucleoprotein(RNP).

In some embodiments of the pharmaceutical composition of the presentapplication, the therapeutic agent (or prophylactic agent) is apolynucleotide, and a molar ratio of nitrogen in the lipid compositionto phosphate in the polynucleotide (N/P ratio) is no more than (about)20:1, no more than (about) 15:1, no more than (about) 10:1, or no morethan (about) 5:1. In some embodiments of the pharmaceutical compositionof the present application, the therapeutic agent (or prophylacticagent) is a polynucleotide, and a molar ratio of nitrogen in the lipidcomposition to phosphate in the polynucleotide (N/P ratio) is no lessthan (about) 20:1, no less than (about) 15:1, no less than (about) 10:1,or no less than (about) 5:1. In some embodiments of the pharmaceuticalcomposition of the present application, the therapeutic agent (orprophylactic agent) is a polynucleotide, and a molar ratio of nitrogenin the lipid composition to phosphate in the polynucleotide (N/P ratio)is from about 5:1 to about 20:1. In some embodiments of thepharmaceutical composition of the present application, the therapeuticagent (or prophylactic agent) is a polynucleotide, and a molar ratio ofnitrogen in the lipid composition to phosphate in the polynucleotide(N/P ratio) is from about 10:1 to about 20:1. In some embodiments of thepharmaceutical composition of the present application, the therapeuticagent (or prophylactic agent) is a polynucleotide, and a molar ratio ofnitrogen in the lipid composition to phosphate in the polynucleotide(N/P ratio) is from about 15:1 to about 20:1. In some embodiments of thepharmaceutical composition of the present application, the therapeuticagent (or prophylactic agent) is a polynucleotide, and a molar ratio ofnitrogen in the lipid composition to phosphate in the polynucleotide(N/P ratio) is from about 5:1 to about 10:1. In some embodiments of thepharmaceutical composition of the present application, the therapeuticagent (or prophylactic agent) is a polynucleotide, and a molar ratio ofnitrogen in the lipid composition to phosphate in the polynucleotide(N/P ratio) is from about 5:1 to about 15:1. In some embodiments of thepharmaceutical composition of the present application, the therapeuticagent (or prophylactic agent) is a polynucleotide, and a molar ratio ofnitrogen in the lipid composition to phosphate in the polynucleotide(N/P ratio) is from about 5:1 to about 20:1. In some embodiments of thepharmaceutical composition of the present application, the therapeuticagent (or prophylactic agent) is a polynucleotide, and a molar ratio ofnitrogen in the lipid composition to phosphate in the polynucleotide(N/P ratio) is from about 15:1 to about 20:1.

In some embodiments of the pharmaceutical composition of the presentapplication, a molar ratio of the therapeutic agent to total lipids ofthe lipid composition is from about 1:1 to about 1:100. In someembodiments of the pharmaceutical composition of the presentapplication, a molar ratio of the therapeutic agent to total lipids ofthe lipid composition is from about 1:1 to about 1:50. In someembodiments of the pharmaceutical composition of the presentapplication, a molar ratio of the therapeutic agent to total lipids ofthe lipid composition is from about 50:1 to about 1:100. In someembodiments of the pharmaceutical composition of the presentapplication, a molar ratio of the therapeutic agent to total lipids ofthe lipid composition is from about 1:1 to about 1:20. In someembodiments of the pharmaceutical composition of the presentapplication, a molar ratio of the therapeutic agent to total lipids ofthe lipid composition is from about 20:1 to about 1:50. In someembodiments of the pharmaceutical composition of the presentapplication, a molar ratio of the therapeutic agent to total lipids ofthe lipid composition is from about 50:1 to about 1:70. In someembodiments of the pharmaceutical composition of the presentapplication, a molar ratio of the therapeutic agent to total lipids ofthe lipid composition is from about 70:1 to about 1:100. In someembodiments of the pharmaceutical composition of the presentapplication, a molar ratio of the therapeutic agent to total lipids ofthe lipid composition is no more than (about) 1:1, no more than (about)1:5, no more than (about) 1:10, no more than (about) 1:15, no more than(about) 1:20, no more than (about) 1:25, no more than (about) 1:30, nomore than (about) 1:35, no more than (about) 1:40, no more than (about)1:45, no more than (about) 1:50, no more than (about) 1:60, no more than(about) 1:70, no more than (about) 1:80, no more than (about) 1:90, ormore than (about) 1:100. In some embodiments of the pharmaceuticalcomposition of the present application, a molar ratio of the therapeuticagent to total lipids of the lipid composition is no less than (about)1:1, no less than (about) 1:5, no less than (about) 1:10, no less than(about) 1:15, no less than (about) 1:20, no less than (about) 1:25, noless than (about) 1:30, no less than (about) 1:35, no less than (about)1:40, no less than (about) 1:45, no less than (about) 1:50, no less than(about) 1:60, no less than (about) 1:70, no less than (about) 1:80, noless than (about) 1:90, or less than (about) 1:100.

In some embodiments of the pharmaceutical composition of the presentapplication, at least (about) 85%, at least (about) 86%, at least(about) 87%, at least (about) 88%, at least (about) 89%, at least(about) 90%, at least (about) 91%, at least (about) 92%, at least(about) 93%, at least (about) 94%, at least (about) 95%, at least(about) 96%, at least (about) 97%, at least (about) 98%, at least(about) 99%, or (about) 100% of the therapeutic agent is encapsulated inparticles of the lipid compositions.

In some embodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particlescharacterized by one or more characteristics of the following: (1) a(e.g., average) size of 100 nanometers (nm) or less; (2) apolydispersity index (PDI) of no more than about 0.2; and (3) a zetapotential of -10 millivolts (mV) to 10 mV. In some embodiments of thepharmaceutical composition of the present application, the lipidcomposition comprises a plurality of particles with a (e.g., average)size from about 50 nanometers (nm) to about 100 nanometers (nm). In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a (e.g., average) size from about 70 nanometers (nm) to about 100nanometers (nm). In some embodiments of the pharmaceutical compositionof the present application, the lipid composition comprises a pluralityof particles with a (e.g., average) size from about 50 nanometers (nm)to about 80 nanometers (nm). In some embodiments of the pharmaceuticalcomposition of the present application, the lipid composition comprisesa plurality of particles with a (e.g., average) size from about 60nanometers (nm) to about 80 nanometers (nm). In some embodiments of thepharmaceutical composition of the present application, the lipidcomposition comprises a plurality of particles with a (e.g., average)size of at most about 100 nanometers (nm), at most about 90 nanometers(nm), at most about 85 nanometers (nm), at most about 80 nanometers(nm), at most about 75 nanometers (nm), at most about 70 nanometers(nm), at most about 65 nanometers (nm), at most about 60 nanometers(nm), at most about 55 nanometers (nm), or at most about 50 nanometers(nm). In some embodiments of the pharmaceutical composition of thepresent application, the lipid composition comprises a plurality ofparticles with a (e.g., average) size of at least about 100 nanometers(nm), at least about 90 nanometers (nm), at least about 85 nanometers(nm), at least about 80 nanometers (nm), at least about 75 nanometers(nm), at least about 70 nanometers (nm), at least about 65 nanometers(nm), at least about 60 nanometers (nm), at least about 55 nanometers(nm), or at least about 50 nanometers (nm). The (e.g., average) size maybe determined by size exclusion chromatography (SEC). The (e.g.,average) size may be determined by spectroscopic method(s) orimage-based method(s), for example, dynamic light scattering, staticlight scattering, multi-angle light scattering, laser light scattering,or dynamic image analysis, or a combination thereof.

In some embodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a polydispersity index (PDI) from about 0.05 to about 0.5. In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a polydispersity index (PDI) from about 0.1 to about 0.5. In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a polydispersity index (PDI) from about 0.1to about 0.3. In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a polydispersity index (PDI) from about 0.2 to about 0.5. In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a polydispersity index (PDI) of no more than about 0.5, no morethan about 0.4, no more than about 0.3, no more than about 0.2, no morethan about 0.1, or no more than about 0.05.

In some embodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a negative zeta potential of -5 millivolts (mV) or less. In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a negative zeta potential of -10 millivolts (mV) or less. In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a negative zeta potential of -15 millivolts (mV) or less. In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a negative zeta potential of -20 millivolts (mV) or less. In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a negative zeta potential of -30 millivolts (mV) or less. In someembodiments, the lipid composition comprises a plurality of particleswith a zeta potential of 0 millivolts (mV) or less. In some embodiments,the lipid composition comprises a plurality of particles with a zetapotential of 5 millivolts (mV) or less. In some embodiments, the lipidcomposition comprises a plurality of particles with a zeta potential of10 millivolts (mV) or less. In some embodiments of the pharmaceuticalcomposition of the present application, the lipid composition comprisesa plurality of particles with a negative zeta potential of 15 millivolts(mV) or less. In some embodiments of the pharmaceutical composition ofthe present application, the lipid composition comprises a plurality ofparticles with a negative zeta potential of 20 millivolts (mV) or less.

In some embodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a negative zeta potential of -5 millivolts (mV) or more. In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a negative zeta potential of -10 millivolts (mV) or more In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a negative zeta potential of -15 millivolts (mV) or more. In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a negative zeta potential of -20 millivolts (mV) or more. In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition comprises a plurality of particleswith a negative zeta potential of -30 millivolts (mV) or more. In someembodiments, the lipid composition comprises a plurality of particleswith a zeta potential of 0 millivolts (mV) or more. In some embodiments,the lipid composition comprises a plurality of particles with a zetapotential of 5 millivolts (mV) or more. In some embodiments, the lipidcomposition comprises a plurality of particles with a zeta potential of10 millivolts (mV) or more. In some embodiments of the pharmaceuticalcomposition of the present application, the lipid composition comprisesa plurality of particles with a zeta potential of 15 millivolts (mV) ormore. In some embodiments of the pharmaceutical composition of thepresent application, the lipid composition comprises a plurality ofparticles with a zeta potential of 20 millivolts (mV) or more.

In some embodiments of the pharmaceutical composition of the presentapplication, the lipid composition has an apparent ionization constant(pKa) outside a range of 6 to 7. In some embodiments of thepharmaceutical composition of the present application, the lipidcomposition has an apparent pKa of about 8 or higher, about 9 or higher,about 10 or higher, about 11 or higher, about 12 or higher, or about 13or higher. In some embodiments of the pharmaceutical composition of thepresent application, the lipid composition has an apparent pKa of about8 to about 13. In some embodiments of the pharmaceutical composition ofthe present application, the lipid composition has an apparent pKa ofabout 8 to about 10. In some embodiments of the pharmaceuticalcomposition of the present application, the lipid composition has anapparent pKa of about 9 to about 11. In some embodiments of thepharmaceutical composition of the present application, the lipidcomposition has an apparent pKa of about 10 to about 13. In someembodiments of the pharmaceutical composition of the presentapplication, the lipid composition has an apparent pKa of about 8 toabout 12. In some embodiments of the pharmaceutical composition of thepresent application, the lipid composition has an apparent pKa of about10 to about 12.

In some embodiments of the pharmaceutical composition of the presentapplication, the SORT lipid in the pharmaceutical composition effects adelivery of the therapeutic agent characterized by one or more of thefollowing: (a) a greater therapeutic effect in a cell of the subjectcompared to that achieved with a reference lipid composition; (b) atherapeutic effect in a greater plurality of cells of the subjectcompared to that achieved with a reference lipid composition; (c) atherapeutic effect in a first plurality of cells of a first cell typeand in a greater second plurality of cells of a second cell type; and(d) a greater therapeutic effect in a first cell of a first cell type ofthe subject compared to that in a second cell of a second cell type ofthe subject. In some embodiments, the first cell type is different fromthe second cell type.

In some embodiments of the pharmaceutical composition of the presentapplication, the cell is a lung cell. In some embodiments, the lung cellis a lung airway cell. Examples of lung airway cells that can betargeted by the delivery of the present application includes but is notlimited to basal cell, secretory cell such as goblet cell and club cell,ciliated cell and any combination thereof. The cells may be located inthe trachea, bronchi, bronchioles, or other parts of the lung orassociated areas.

In some embodiments of the pharmaceutical composition of the presentapplication, the therapeutic effect is characterized by atherapeutically effective amount of the therapeutic agent, for example,in a lung, a lung cell, a plurality of lung cells, or a lung cell typeof the subject. In some embodiments, the therapeutic effect ischaracterized by an activity of the therapeutic agent, for example, in alung, a lung cell, a plurality of lung cells, or a lung cell type of thesubject. In some embodiments, the therapeutic effect is characterized byan effect of the therapeutic agent, for example, in a lung, a lung cell,a plurality of lung cells, or a lung cell type of the subject. In someembodiments, the greater therapeutic effect is characterized by agreater therapeutic amount of the therapeutic agent. In someembodiments, the greater therapeutic effect is characterized by agreater activity of the therapeutic agent. In some embodiments, thegreater therapeutic effect is characterized by a greater effect of thetherapeutic agent.

In some embodiments of the pharmaceutical composition of the presentapplication, the SORT lipid in the pharmaceutical composition effectsdelivery of the therapeutic agent to the cell of the subjectcharacterized by a greater therapeutic effect compared to that achievedwith a reference lipid composition. In some embodiments, the referencelipid composition does not comprise the SORT lipid. In some embodiments,the reference lipid composition does not comprise the amount of the SORTlipid. In some embodiments, the reference lipid comprises13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol(“LF92”), a phospholipid, cholesterol, and a PEG-lipid.

In some embodiments of the pharmaceutical composition of the presentapplication, the SORT lipid in the pharmaceutical composition achievesabout 1.1-fold to about 20-fold therapeutic effect compared to thatachieved with a reference lipid composition. In some embodiments, theSORT lipid achieves about 1.1-fold to about 10-fold therapeutic effectcompared to that achieved with a reference lipid composition. In someembodiments, the SORT lipid achieves about 5-fold to about 10-foldtherapeutic effect compared to that achieved with a reference lipidcomposition. In some embodiments, the SORT lipid achieves about 10-foldto about 20-fold therapeutic effect compared to that achieved with areference lipid composition. In some embodiments, the SORT lipidachieves at least about 1.1-fold, at least about 1.5-fold, at leastabout 2-fold, at least about 3-fold, at least about 4-fold, at leastabout 5-fold, at least about 6-fold, at least about 7-fold, at leastabout 8-fold, at least about 9-fold, at least about 10-fold, at leastabout 11-fold, at least about 12-fold, at least about 13-fold, at leastabout 14-fold, at least about 15-fold, at least about 16-fold, at leastabout 17-fold, at least about 18-fold, at least about 19-fold, or atleast about 20-fold therapeutic effect compared to that achieved with areference lipid composition.

In some embodiments of the pharmaceutical composition of the presentapplication, the SORT lipid in the pharmaceutical composition achievesabout 1.1-fold to about 20-fold greater therapeutic effect compared tothat achieved with a reference lipid composition in cells selected frombasal cell, secretory cell such as goblet cell and club cell, ciliatedcell and any combination thereof. In some embodiments, the SORT lipidachieves about 1.1-fold to about 10-fold therapeutic effect compared tothat achieved with a reference lipid composition in cells selected frombasal cell, secretory cell such as goblet cell and club cell, ciliatedcell and any combination thereof. In some embodiments, the SORT lipidachieves about 5-fold to about 10-fold greater therapeutic effectcompared to that achieved with a reference lipid composition in cellsselected from basal cell, secretory cell such as goblet cell and clubcell, ciliated cell and any combination thereof. In some embodiments,the SORT lipid achieves about 10-fold to about 20-fold greatertherapeutic effect compared to that achieved with a reference lipidcomposition in cells selected from basal cell, secretory cell such asgoblet cell and club cell, ciliated cell and any combination thereof. Insome embodiments, the SORT lipid achieves at least about 1.1-fold, atleast about 1.5-fold, at least about 2-fold, at least about 3-fold, atleast about 4-fold, at least about 5-fold, at least about 6-fold, atleast about 7-fold, at least about 8-fold, at least about 9-fold, atleast about 10-fold, at least about 11-fold, at least about 12-fold, atleast about 13-fold, at least about 14-fold, at least about 15-fold, atleast about 16-fold, at least about 17-fold, at least about 18-fold, atleast about 19-fold, or at least about 20-fold therapeutic effectcompared to that achieved with a reference lipid composition in cellsselected from basal cell, secretory cell such as goblet cell and clubcell, ciliated cell and any combination thereof.

In some embodiments of the pharmaceutical composition of the presentapplication, the SORT lipid in the pharmaceutical composition effectsdelivery of the therapeutic agent to cells of the subject characterizedby a therapeutic effect in a greater plurality of cells compared to thatachieved with a reference lipid composition. In some embodiments, thereference lipid composition does not comprise the SORT lipid. In someembodiments, the reference lipid composition does not comprise theamount of the SORT lipid. In some embodiments, the reference lipidcomprises13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol(“LF92”), a phospholipid, cholesterol, and a PEG-lipid.

In some embodiments of the pharmaceutical composition of the presentapplication, the SORT lipid in the pharmaceutical composition achievestherapeutic effect in about 1.1-fold to about 20-fold cells compared tothat achieved with a reference lipid composition. In some embodiments,the SORT lipid achieves therapeutic effect in about 1.1-fold to about10-fold cells compared to that achieved with a reference lipidcomposition. In some embodiments, the SORT lipid achieves therapeuticeffect in about 5-fold to about 10-fold cells compared to that achievedwith a reference lipid composition. In some embodiments, the SORT lipidachieves therapeutic effect in about 10-fold to about 20-fold cellscompared to that achieved with a reference lipid composition. In someembodiments, the SORT lipid achieves therapeutic effect in at leastabout 1.1-fold, at least about 1.5-fold, at least about 2-fold, at leastabout 3-fold, at least about 4-fold, at least about 5-fold, at leastabout 6-fold, at least about 7-fold, at least about 8-fold, at leastabout 9-fold, at least about 10-fold, at least about 11-fold, at leastabout 12-fold, at least about 13-fold, at least about 14-fold, at leastabout 15-fold, at least about 16-fold, at least about 17-fold, at leastabout 18-fold, at least about 19-fold, or at least about 20-fold cellscompared to that achieved with a reference lipid composition.

In some embodiments of the pharmaceutical composition of the presentapplication, the SORT lipid in the pharmaceutical composition achievestherapeutic effect in about 1.1-fold to about 20-fold cells compared tothat achieved with a reference lipid composition, wherein the cells isselected from basal cell, secretory cell such as goblet cell and clubcell, ciliated cell and any combination thereof. In some embodiments,the SORT lipid achieves therapeutic effect in about 1.1-fold to about10-fold more cells compared to that achieved with a reference lipidcomposition, wherein the cells are selected from basal cell, secretorycell such as goblet cell and club cell, ciliated cell and anycombination thereof. In some embodiments, the SORT lipid achievestherapeutic effect in about 5-fold to about 10-fold more cells comparedto that achieved with a reference lipid composition, wherein the cellsare selected from basal cell, secretory cell such as goblet cell andclub cell, ciliated cell and any combination thereof. In someembodiments, the SORT lipid achieves therapeutic effect in about 10-foldto about 20-fold more cells compared to that achieved with a referencelipid composition, wherein the cells are selected from basal cell,secretory cell such as goblet cell and club cell, ciliated cell and anycombination thereof. In some embodiments, the SORT lipid achievestherapeutic effect in about 1.1-fold, at least about 1.5-fold, at leastabout 2-fold, at least about 3-fold, at least about 4-fold, at leastabout 5-fold, at least about 6-fold, at least about 7-fold, at leastabout 8-fold, at least about 9-fold, at least about 10-fold, at leastabout 11-fold, at least about 12-fold, at least about 13-fold, at leastabout 14-fold, at least about 15-fold, at least about 16-fold, at leastabout 17-fold, at least about 18-fold, at least about 19-fold, or atleast about 20-fold more cells compared to that achieved with areference lipid composition, wherein the cells are selected from basalcell, secretory cell such as goblet cell and club cell, ciliated celland any combination thereof.

In some embodiments of the pharmaceutical composition of the presentapplication, the SORT lipid in the pharmaceutical composition effectsdelivery of the therapeutic agent to cells of the subject characterizedby a therapeutic effect in a first plurality of cells of a first celltype and in a greater therapeutic effect in a second plurality of cellsof a second cell type. In some embodiments, the first cell type isdifferent from the second cell type.

In some embodiments of the pharmaceutical composition of the presentapplication, the first cell type is a lung cell. In some embodiments,the first cell type is a lung airway cell. Examples of lung airway cellthat can be targeted by the delivery of the present application includesbut is not limited to basal cell, secretory cell such as goblet cell andclub cell, ciliated cell and any combination thereof.

In some embodiments of the pharmaceutical composition of the presentapplication, the second cell type is a lung cell. In some embodiments,the second cell type is a lung airway cell. Examples of lung airwaycells that can be targeted by the delivery of the present applicationincludes but is not limited to basal cell, secretory cell such as gobletcell and club cell, ciliated cell and any combination thereof. The cellsmay be located in the trachea, bronchi, bronchioles, or other parts ofthe lung or associated areas.

In some embodiments of the pharmaceutical composition of the presentapplication, the SORT lipid in the pharmaceutical composition achievestherapeutic effect in about 1.1-fold to about 20-fold greater secondplurality of cells of the second cell type compared to the firstplurality of cells of the first cell type. In some embodiments, the SORTlipid achieves therapeutic effect in about 1.1-fold to about 10-foldgreater second plurality of cells of the second cell type compared tothe first plurality of cells of the first cell type. In someembodiments, the SORT lipid achieves therapeutic effect in about 5-foldto about 10-fold greater second plurality of cells of the second celltype compared to the first plurality of cells of the first cell type. Insome embodiments, the SORT lipid achieves therapeutic effect in about10-fold to about 20-fold greater second plurality of cells of the secondcell type compared to the first plurality of cells of the first celltype. In some embodiments, the SORT lipid achieves therapeutic effect inat least about 1.1-fold, at least about 1.5-fold, at least about 2-fold,at least about 3-fold, at least about 4-fold, at least about 5-fold, atleast about 6-fold, at least about 7-fold, at least about 8-fold, atleast about 9-fold, at least about 10-fold, at least about 11-fold, atleast about 12-fold, at least about 13-fold, at least about 14-fold, atleast about 15-fold, at least about 16-fold, at least about 17-fold, atleast about 18-fold, at least about 19-fold, or at least about 20-foldgreater second plurality of cells of the second cell type compared tothe first plurality of cells of the first cell type.

In some embodiments of the pharmaceutical composition of the presentapplication, the SORT lipid in the pharmaceutical composition effectsdelivery of the therapeutic agent to cells of the subject characterizedby a greater therapeutic effect in a first cell of a first cell typecompared to that in a second cell of a second cell type. In someembodiments, the first cell type is different from the second cell type.

In some embodiments of the pharmaceutical composition of the presentapplication, the first cell type is a lung cell. In some embodiments,the first cell type is a lung airway cell. Examples of lung airway cellsthat can be targeted by the delivery of the present application includesbut is not limited to basal cell, secretory cell such as goblet cell andclub cell, ciliated cell and any combination thereof. The cells may belocated in the trachea, bronchi, bronchioles, or other parts of the lungor associated areas.

In some embodiments of the pharmaceutical composition of the presentapplication, the second cell type is a lung cell. In some embodiments,the second cell type is a lung airway cell. Examples of lung airway cellthat can be targeted by the delivery of the present application includesbut is not limited to basal cell, secretory cell such as goblet cell andclub cell, ciliated cell and any combination thereof.

In some embodiments of the pharmaceutical composition of the presentapplication, the SORT lipid in the pharmaceutical composition achievesabout 1.1-fold to about 20-fold therapeutic effect in first cell of thefirst cell type compared to that achieved in the second cell of thesecond cell type. In some embodiments, the SORT lipid achieves about1.1-fold to about 10-fold therapeutic effect in first cell of the firstcell type compared to that achieved in the second cell of the secondcell type. In some embodiments, the SORT lipid achieves about 5-fold toabout 10-fold therapeutic effect in first cell of the first cell typecompared to that achieved in the second cell of the second cell type. Insome embodiments, the SORT lipid achieves about 10-fold to about 20-foldtherapeutic effect in first cell of the first cell type compared to thatachieved in the second cell of the second cell type. In some embodimentsof the method, the SORT lipid achieves at least about 1.1-fold, at leastabout 1.5-fold, at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 6-fold, at leastabout 7-fold, at least about 8-fold, at least about 9-fold, at leastabout 10-fold, at least about 11-fold, at least about 12-fold, at leastabout 13-fold, at least about 14-fold, at least about 15-fold, at leastabout 16-fold, at least about 17-fold, at least about 18-fold, at leastabout 19-fold, or at least about 20-fold therapeutic effect in firstcell of the first cell type compared to that achieved in the second cellof the second cell type.

In some embodiments, the pharmaceutical composition is an aerosolcomposition. In some embodiments, the aerosol composition is generatedby a nebulizer at a nebulization rate of no more than 70 mL/minute. Insome embodiments, the aerosol composition is generated by a nebulizer ata nebulization rate of no more than 50 mL/minute. In some embodiments,the aerosol composition is generated by a nebulizer at a nebulizationrate of no more than 30 mL/minute.

In some embodiments, the aerosol composition has an average droplet sizefrom about to about 0.5 micron (µm) to about 10 µm. In some embodiments,the aerosol composition has an average droplet size from about to about0.5 micron (µm) to about 10 µm. In some embodiments, the aerosolcomposition has an average droplet size from about to about 1 micron(µm) to about 10 µm. In some embodiments, the aerosol composition has anaverage droplet size from about to about 0.5 micron (µm) to about 5 µm.In some embodiments, the aerosol droplets are generated by a nebulizerat a nebulization rate of no more than 70 mL/minute. In someembodiments, the aerosol droplets have a mass median aerodynamicdiameter (MMAD) from about 0.5 micron (µm) to about 10 µm. In someembodiments, the droplet size varies less than about 50% for a durationof about 24 hours under a storage condition. In some embodiments,droplets of said aerosol composition are characterized by a geometricstandard deviation (GSD) of no more than about 3.

In some embodiments, provided herein are (e.g., pharmaceutical)compositions that comprise components that allow for an improvedefficacy or outcome based on the delivery of the polynucleotide. Thecompositions described elsewhere herein may be more effective atdelivery to a particular cell, cell type, organ, or bodily region ascompared to a reference composition or compound. The compositionsdescribed elsewhere herein may be more effective at generating increaseexpression of a corresponding polypeptide of a delivered polynucleotide.The compositions described elsewhere herein may be more effective atgenerating a larger number of cells that express a correspondingpolypeptide of a delivered polynucleotide. The compositions describedelsewhere herein may result in an increase uptake of the polynucleotideas compared to a reference polynucleotide. The increased uptake may beresult of improved stability of polynucleotide or an improved targetingof the composition to a particular cell type or organ. In someembodiments, the SORT lipid is present in an amount in the lipidcomposition to effect a greater expression or activity of thepolynucleotide (or corresponding polypeptide of the polynucleotide) in acell compared to that achieved with a reference lipid compositioncomprising13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol(“LF92”), a phospholipid, cholesterol, and a PEG-lipid. In someembodiments, the SORT lipid is present in an amount in the lipidcomposition to effect at least a 1.1 fold greater expression or activityof the polynucleotide (or corresponding polypeptide of thepolynucleotide) in a cell compared to that achieved with a referencelipid composition comprising LF92, a phospholipid, cholesterol, and aPEG-lipid. In some embodiments, the SORT lipid is present in an amountin the lipid composition to effect at least a 2-fold greater expressionor activity of the polynucleotide (or corresponding polypeptide of thepolynucleotide) in a cell compared to that achieved with a referencelipid composition comprising LF92, a phospholipid, cholesterol, and aPEG-lipid. In some embodiments, the SORT lipid is present in an amountin the lipid composition to effect at least a 5 fold greater expressionor activity of the polynucleotide (or corresponding polypeptide of thepolynucleotide) in a cell compared to that achieved with a referencelipid composition comprising LF92, a phospholipid, cholesterol, and aPEG-lipid. In some embodiments, the SORT lipid is present in an amountin the lipid composition to effect at least a 10- fold greaterexpression or activity of the polynucleotide (or correspondingpolypeptide of the polynucleotide) in a cell compared to that achievedwith a reference lipid composition comprising LF92, a phospholipid,cholesterol, and a PEG-lipid.

In some embodiments, the SORT lipid is present in an amount in the lipidcomposition to effect an expression or activity of the polynucleotide(or corresponding polypeptide of the polynucleotide) in a greaterplurality of cells compared to that achieved with a reference lipidcomposition comprising LF92, a phospholipid, cholesterol, and aPEG-lipid. In some embodiments, the SORT lipid is present in an amountin the lipid composition to effect an expression or activity of thepolynucleotide (or corresponding polypeptide of the polynucleotide) inat least a 1.1-fold greater plurality of cells compared to that achievedwith a reference lipid composition comprising LF92, a phospholipid,cholesterol, and a PEG-lipid. In some embodiments, the SORT lipid ispresent in an amount in the lipid composition to effect an expression oractivity of the polynucleotide (or corresponding polypeptide of thepolynucleotide) in at least a 2-fold greater plurality of cells comparedto that achieved with a reference lipid composition comprising LF92, aphospholipid, cholesterol, and a PEG-lipid. In some embodiments, theSORT lipid is present in an amount in the lipid composition to effect anexpression or activity of the polynucleotide (or correspondingpolypeptide of the polynucleotide) in at least a 5-fold greaterplurality of cells compared to that achieved with a reference lipidcomposition comprising LF92, a phospholipid, cholesterol, and aPEG-lipid. In some embodiments, the SORT lipid is present in an amountin the lipid composition to effect an expression or activity of thepolynucleotide (or corresponding polypeptide of the polynucleotide) inat least a 10-fold greater plurality of cells compared to that achievedwith a reference lipid composition comprising LF92, a phospholipid,cholesterol, and a PEG-lipid.

In some embodiments, the SORT lipid is present in an amount in the lipidcomposition to effect an uptake of the polynucleotide in a greaterplurality of cells compared to that achieved with a reference lipidcomposition comprising LF92, a phospholipid, cholesterol, and aPEG-lipid. In some embodiments, the SORT lipid is present in an amountin the lipid composition to effect an uptake of the polynucleotide in agreater amount to a cell compared to that achieved with a referencelipid composition comprising LF92, a phospholipid, cholesterol, and aPEG-lipid.

METHODS

In some embodiments, provided herein in some embodiments include amethod for delivery by nebulization to lung cell(s) of a subject, themethod comprising: administering to said subject a (e.g.,pharmaceutical) composition (such as one described herein) comprising atherapeutic agent assembled with a lipid composition, which lipidcomposition (such as one described herein) comprises: (i) an ionizablecationic lipid (such as one described herein); and (ii) a selectiveorgan targeting (SORT) lipid (such as one described herein) separatefrom said ionizable cationic lipid, thereby delivering said therapeuticagent to said lung cell(s) of a lung of said subject. In someembodiments, the method provides a (e.g., therapeutically) effectiveamount or activity of said therapeutic agent in at least about 5%, 10%,15%, or 20% lung epithelial cells of said subject. In some embodiments,the method provides a (e.g., therapeutically) effective amount oractivity of said therapeutic agent in at least about 2%, 5%, or 10% lungciliated cells of said subject. In some embodiments, the method providesa (e.g., therapeutically) effective amount or activity of saidtherapeutic agent in at least about 5%, 10%, 15%, or 20% lung secretorycells of said subject. In some embodiments, the method provides a (e.g.,therapeutically) effective amount or activity of said therapeutic agentin at least about 5%, 10%, 15%, or 20% lung club cells of said subject.In some embodiments, the method provides a (e.g., therapeutically)effective amount or activity of said therapeutic agent in at least about5%, 10%, 15%, or 20% lung goblet cells of said subject. In someembodiments, the method provides a (e.g., therapeutically) effectiveamount or activity of said therapeutic agent in at least about 5%, 10%,15%, or 20% lung basal cells of said subject. In some embodiments, thelipid composition comprises a phospholipid. In some embodiments, the(e.g., pharmaceutical) composition comprising said therapeutic agentassembled with said lipid composition is an aerosol composition (such asone described herein).

In some embodiments, provided herein is a method for potent delivery toa cell of a subject comprising administrating to the subject thepharmaceutical composition as described in the present application. Insome embodiments of the method, the pharmaceutical composition comprisesa therapeutic agent (or prophylactic agent) assembled with a lipidcomposition as described in the present application, wherein the lipidcomposition comprises (i) an ionizable cationic lipid; and (iii) aselective organ targeting (SORT) lipid separate from the ionizablecationic lipid. The lipid composition may further comprise aphospholipid.

In some embodiments of the method, the cell is a lung cell. In someembodiments, the lung cell is a lung airway cell. Examples of lungairway cells that can be targeted by the delivery of the presentapplication includes but is not limited to basal cell, secretory cellsuch as goblet cell and club cell, ciliated cell and any combinationthereof.

In some embodiments of the method, the SORT lipid effects delivery ofthe therapeutic agent to the cell of the subject characterized by agreater therapeutic effect compared to that achieved with a referencelipid composition. In some embodiments, the reference lipid compositiondoes not comprise the SORT lipid. In some embodiments, the referencelipid composition does not comprise the amount of the SORT lipid. Insome embodiments, the reference lipid comprises13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol(“LF92”), a phospholipid, cholesterol, and a PEG-lipid.

In some embodiments of the method, the SORT lipid achieves about1.1-fold to about 20-fold therapeutic effect compared to that achievedwith a reference lipid composition. In some embodiments of the method,the SORT lipid achieves about 1.1-fold to about 10-fold therapeuticeffect compared to that achieved with a reference lipid composition. Insome embodiments of the method, the SORT lipid achieves about 1.1-foldto about 5-fold therapeutic effect compared to that achieved with areference lipid composition. In some embodiments of the method, the SORTlipid achieves about 5-fold to about 10-fold therapeutic effect comparedto that achieved with a reference lipid composition. In some embodimentsof the method, the SORT lipid achieves about 10-fold to about 20-foldtherapeutic effect compared to that achieved with a reference lipidcomposition. In some embodiments of the method, the SORT lipid achievesat least about 1.1-fold, at least about 1.5-fold, at least about 2-fold,at least about 3-fold, at least about 4-fold, at least about 5-fold, atleast about 6-fold, at least about 7-fold, at least about 8-fold, atleast about 9-fold, at least about 10-fold, at least about 11-fold, atleast about 12-fold, at least about 13-fold, at least about 14-fold, atleast about 15-fold, at least about 16-fold, at least about 17-fold, atleast about 18-fold, at least about 19-fold, or at least about 20-foldtherapeutic effect compared to that achieved with a reference lipidcomposition.

In some embodiments of the method, the SORT lipid achieves about1.1-fold to about 20-fold therapeutic effect compared to that achievedwith a reference lipid composition in cells selected from basal cell,secretory cell such as goblet cell and club cell, ciliated cell and anycombination thereof. In some embodiments of the method, the SORT lipidachieves about 1.1-fold to about 10-fold greater therapeutic effectcompared to that achieved with a reference lipid composition in cellsselected from basal cell, secretory cell such as goblet cell and clubcell, ciliated cell and any combination thereof. In some embodiments ofthe method, the SORT lipid achieves about 1.1-fold to about 5-foldgreater therapeutic effect compared to that achieved with a referencelipid composition in cells selected from basal cell, secretory cell suchas goblet cell and club cell, ciliated cell and any combination thereof.In some embodiments of the method, the SORT lipid achieves about 10-foldto about 20-fold greater therapeutic effect compared to that achievedwith a reference lipid composition in cells selected from basal cell,secretory cell such as goblet cell and club cell, ciliated cell and anycombination thereof. In some embodiments of the method, the SORT lipidachieves at least about 1.1-fold, at least about 1.5-fold, at leastabout 2-fold, at least about 3-fold, at least about 4-fold, at leastabout 5-fold, at least about 6-fold, at least about 7-fold, at leastabout 8-fold, at least about 9-fold, at least about 10-fold, at leastabout 11-fold, at least about 12-fold, at least about 13-fold, at leastabout 14-fold, at least about 15-fold, at least about 16-fold, at leastabout 17-fold, at least about 18-fold, at least about 19-fold, or atleast about 20-fold therapeutic effect compared to that achieved with areference lipid composition in cells selected from basal cell, secretorycell such as goblet cell and club cell, ciliated cell and anycombination thereof.

In some embodiments of the method, the SORT lipid effects delivery ofthe therapeutic agent to cells of the subject characterized by atherapeutic effect in a greater plurality of cells compared to thatachieved with a reference lipid composition. In some embodiments, thereference lipid composition does not comprise the SORT lipid. In someembodiments, the reference lipid composition does not comprise theamount of the SORT lipid. In some embodiments, the reference lipidcomprises13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol(“LF92”), a phospholipid, cholesterol, and a PEG-lipid.

In some embodiments of the method, the SORT lipid achieves therapeuticeffect in about 1.1-fold to about 20-fold cells compared to thatachieved with a reference lipid composition. In some embodiments of themethod, the SORT lipid achieves therapeutic effect in about 1.1-fold toabout 10-fold cells compared to that achieved with a reference lipidcomposition. In some embodiments of the method, the SORT lipid achievestherapeutic effect in about 1.1-fold to about 5-fold cells compared tothat achieved with a reference lipid composition. In some embodiments ofthe method, the SORT lipid achieves therapeutic effect in about 10-foldto about 20-fold cells compared to that achieved with a reference lipidcomposition. In some embodiments of the method, the SORT lipid achievestherapeutic effect in at least about 1.1-fold, at least about 5-fold, atleast about 10-fold, at least about 20-fold, at least about 50-fold, orat least about 100-fold cells compared to that achieved with a referencelipid composition.

In some embodiments of the method, the SORT lipid achieves therapeuticeffect in about 1.1-fold to about 20-fold cells compared to thatachieved with a reference lipid composition, wherein the cells areselected from basal cell, secretory cell such as goblet cell and clubcell, ciliated cell and any combination thereof. In some embodiments ofthe method, the SORT lipid achieves therapeutic effect in about 1.1-foldto about 10-fold cells compared to that achieved with a reference lipidcomposition, wherein the cells are selected from basal cell, secretorycell such as goblet cell and club cell, ciliated cell and anycombination thereof. In some embodiments of the method, the SORT lipidachieves therapeutic effect in about 5-fold to about 10-fold more cellscompared to that achieved with a reference lipid composition, whereinthe cells are selected from basal cell, secretory cell such as gobletcell and club cell, ciliated cell and any combination thereof. In someembodiments of the method, the SORT lipid achieves therapeutic effect inabout 10-fold to about 20-fold more cells compared to that achieved witha reference lipid composition, wherein the cells are selected from basalcell, secretory cell such as goblet cell and club cell, ciliated celland any combination thereof. In some embodiments of the method, the SORTlipid achieves therapeutic effect in at least about 1.1-fold, at leastabout 1.5-fold, at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 6-fold, at leastabout 7-fold, at least about 8-fold, at least about 9-fold, at leastabout 10-fold, at least about 11-fold, at least about 12-fold, at leastabout 13-fold, at least about 14-fold, at least about 15-fold, at leastabout 16-fold, at least about 17-fold, at least about 18-fold, at leastabout 19-fold, or at least about 20-fold more cells compared to thatachieved with a reference lipid composition, wherein the cells isselected from basal cell, secretory cell such as goblet cell and clubcell, ciliated cell and any combination thereof.

In some embodiments of the method, the pharmaceutical composition of thepresent application can be administrated through any suitable routesincluding, for example, oral, rectal, vaginal, transmucosal, pulmonaryincluding intratracheal or inhaled, or intestinal administration;parenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

In some embodiments of the method, the pharmaceutical composition of thepresent application can be administered in a local rather than systemicmanner, for example, via injection of the pharmaceutical compositiondirectly into a targeted tissue, preferably in a sustained releaseformulation. Local delivery can be affected in various ways, dependingon the tissue to be targeted.

In some embodiments of the method, aerosols containing the compositionof the present application can be inhaled (for nasal, tracheal, orbronchial delivery). In some embodiments, the composition of the presentapplication can be injected into the site of injury, diseasemanifestation, or pain, for example. In some embodiments, thecomposition of the present application can be provided in lozenges fororal, tracheal, or esophageal application. In some embodiments, thecomposition of the present application can be supplied in liquid, tabletor capsule form for administration to the stomach or intestines. In someembodiments, the composition of the present application can be suppliedin suppository form for rectal or vaginal application. In someembodiments, the composition of the present application can even bedelivered to the eye by use of creams, drops, or even injection.

In another aspect, provided herein is a method for targeted delivery tocells of a subject, comprising administrating to the subject thepharmaceutical composition as described in the present application. Insome embodiments of the method, the pharmaceutical composition comprisesa therapeutic agent (or prophylactic agent) assembled with a lipidcomposition as described in the present application, wherein the lipidcomposition comprises (i) an ionizable cationic lipid; and (ii) aselective organ targeting (SORT) lipid separate from the ionizablecationic lipid. The lipid composition may further comprise aphospholipid.

In some embodiments of the method, the SORT lipid effects delivery ofthe therapeutic agent to a greater proportion of cell types as comparedto that achieved with a reference lipid composition. In someembodiments, the reference lipid composition does not comprise the SORTlipid. In some embodiments, the reference lipid composition does notcomprise the amount of the SORT lipid. In some embodiments, thereference lipid comprises13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol(“LF92”), a phospholipid, cholesterol, and a PEG-lipid.

In some embodiments of the method, the cell is a lung cell. In someembodiments, the lung cell is a lung airway cell. Examples of lungairway cells that can be targeted by the delivery of the presentapplication includes but is not limited to basal cell, secretory cellsuch as goblet cell and club cell, ciliated cell and any combinationthereof.

In some embodiments of the method, the SORT lipid effects delivery ofthe therapeutic agent to cells of the subject characterized by atherapeutic effect in a first plurality of cells of a first cell typeand in a greater therapeutic effect in a second plurality of cells of asecond cell type. In some embodiments, the first cell type is differentfrom the second cell type.

In some embodiments of the method, the first cell type is a lung cell.In some embodiments, the first cell type is a lung airway cell. Examplesof lung airway cells that can be targeted by the delivery of the presentapplication includes but is not limited to basal cell, secretory cellsuch as goblet cell and club cell, ciliated cell and any combinationthereof.

In some embodiments of the method, the second cell type is a lung cell.In some embodiments, the second cell type is a lung airway cell.Examples of lung airway cells that can be targeted by the delivery ofthe present application includes but is not limited to basal cell,secretory cell such as goblet cell and club cell, ciliated cell and anycombination thereof.

In some embodiments of the method, the SORT lipid achieves therapeuticeffect in about 1.1-fold to about 20-fold greater second plurality ofcells of the second cell type compared to the first plurality of cellsof the first cell type. In some embodiments of the method, the SORTlipid achieves therapeutic effect in about 1.1-fold to about 10-foldgreater second plurality of cells of the second cell type compared tothe first plurality of cells of the first cell type. In some embodimentsof the method, the SORT lipid achieves therapeutic effect in about1.1-fold to about 5-fold greater second plurality of cells of the secondcell type compared to the first plurality of cells of the first celltype. In some embodiments of the method, the SORT lipid achievestherapeutic effect in about 10-fold to about 20-fold greater secondplurality of cells of the second cell type compared to the firstplurality of cells of the first cell type. In some embodiments of themethod, the SORT lipid achieves therapeutic effect in at least about1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about3-fold, at least about 4-fold, at least about 5-fold, at least about6-fold, at least about 7-fold, at least about 8-fold, at least about9-fold, at least about 10-fold, at least about 11-fold, at least about12-fold, at least about 13-fold, at least about 14-fold, at least about15-fold, at least about 16-fold, at least about 17-fold, at least about18-fold, at least about 19-fold, or at least about 20-fold greatersecond plurality of cells of the second cell type compared to the firstplurality of cells of the first cell type.

In some embodiments of the method, the SORT lipid effects delivery ofthe therapeutic agent to cells of the subject characterized by a greatertherapeutic effect in a first cell of a first cell type compared to thatin a second cell of a second cell type. In some embodiments, the firstcell type is different from the second cell type.

In some embodiments of the method, the first cell type is a lung cell.In some embodiments, the first cell type is a lung airway cell. Examplesof lung airway cells that can be targeted by the delivery of the presentapplication includes but is not limited to basal cell, secretory cellsuch as goblet cell and club cell, ciliated cell and any combinationthereof.

In some embodiments of the method, the second cell type is a lung cell.In some embodiments, the second cell type is a lung airway cell.Examples of lung airway cells that can be targeted by the delivery ofthe present application includes but is not limited to basal cell,secretory cell such as goblet cell and club cell, ciliated cell and anycombination thereof.

In some embodiments of the method, the SORT lipid achieves about1.1-fold to about 20-fold therapeutic effect in first cell of the firstcell type compared to that achieved in the second cell of the secondcell type. In some embodiments of the method, the SORT lipid achievesabout 1.1-fold to about 10-fold therapeutic effect in first cell of thefirst cell type compared to that achieved in the second cell of thesecond cell type. In some embodiments of the method, the SORT lipidachieves about 5-fold to about 10-fold therapeutic effect in first cellof the first cell type compared to that achieved in the second cell ofthe second cell type. In some embodiments of the method, the SORT lipidachieves about 10-fold to about 20-fold therapeutic effect in first cellof the first cell type compared to that achieved in the second cell ofthe second cell type. In some embodiments of the method, the SORT lipidachieves at least about 1.1-fold, at least about 1.5-fold, at leastabout 2-fold, at least about 3-fold, at least about 4-fold, at leastabout 5-fold, at least about 6-fold, at least about 7-fold, at leastabout 8-fold, at least about 9-fold, at least about 10-fold, at leastabout 11-fold, at least about 12-fold, at least about 13-fold, at leastabout 14-fold, at least about 15-fold, at least about 16-fold, at leastabout 17-fold, at least about 18-fold, at least about 19-fold, or atleast about 20-fold therapeutic effect in first cell of the first celltype compared to that achieved in the second cell of the second celltype.

In some embodiments, the delivery of the therapeutic to a cell may alterthe genome, transcriptome, or expression levels. The cell may be allowedto, or able to, propagate and the alteration may be passed on to thecells generated from the cell that the therapeutic was delivered to. Inthis manner, the therapeutic effect may be propagated to a larger numberof cells. The alteration to the genome, transcriptome or expressionlevel may also persist in a given cell.

Basal Cells

Basal cells are derived from undifferentiated columnar epithelium in thedeveloping airway. They are characterized by basal position in thecolumnar epithelium, the presence of hemidesmosomes (characterized byalpha 6 beta 4 integrins), cytokeratins 5 and 14, and the nuclearprotein p63. The distribution of basal cells varies by airway level andanimal species. Airways that are larger in diameter have more basalcells than airways with smaller diameters. As the airway decreases indiameter, the number of basal cells also decreases, and none are presentin the terminal bronchioles.

In another aspect, provided herein is a method for delivery to basalcells of a subject, comprising administrating to the subject thepharmaceutical composition as described in the present application. Insome embodiments of the method, the pharmaceutical composition comprisesa therapeutic agent (or prophylactic agent) assembled with a lipidcomposition as described in the present application, wherein the lipidcomposition comprises (i) an ionizable cationic lipid; and (ii) aselective organ targeting (SORT) lipid separate from the ionizablecationic lipid. The lipid composition may further comprise aphospholipid. In some embodiments, the basal cell is a lung basal cell.

In some embodiments of the method, the method delivers the therapeuticagent to an organ or tissue of the subject to result in a therapeuticeffect detectable in basal cells in the organ or tissue of the subject.In some embodiments, the method delivers the therapeutic agent to anorgan or tissue of the subject to result in a therapeutic effectdetectable in at least about 5%, at least about 10%, at least about 15%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50% basal cells in the organ or tissue of the subject.

In some embodiments of the method, the organ is lung. In someembodiments, the tissue is lung tissue. In some embodiment, the tissueis lung airway tissue. In some embodiments, the method delivers thetherapeutic agent to the subject’s lung to result in a therapeuticeffect detectable in at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 30%, at least about 40%,at least about 50% basal cells in the subject’s lung.

In some embodiments of the method, the pharmaceutical composition isadministrated to the subject through any suitable delivery. In someembodiment, the pharmaceutical composition is administrated to thesubject through inhalation. In some embodiments, the pharmaceuticalcomposition is administrated to the subject through systemicadministration such as intravenous administration.

Ciliated Cells

Ciliated cells are those cells with cilia structures on the cellsurface. Examples of ciliated cells include but are not limited torespiratory tract ciliated cells, oviduct ciliated cell, uterineendometrial ciliated cells, rete testis ciliated cells, ductulusefferens ciliated cells, and/or ciliated ependymal cells. Humanrespiratory tract ciliated cells bear 200 to 300 cilia on their surface.Cilia are elongated motile cylindrical projections from the apical cellmembrane, approximately 0.25 mm in diameter, that contain microtubulesand cytoplasm in continuity with that of the cell. Human tracheal ciliaare 5 to 8 mm long, becoming shorter in more distal airways.

The structure of a cilium is complex and consists of an axoneme,anchored by a basal body and a rootlet to the cell, and it possessessome smaller claw-like formations on its tip. The direction in which thebasal body points defines the orientation of the cilium and thedirection of the effective beat. The axoneme contains nine pairs ofmicrotubules which surround a central pair of microtubules, as well asradial spokes and peripheral nexin links, which to a great extentmaintain the wheel-like arrangement of the cilium. Inner and outer armsattach to the microtubules. The main structural protein of the doubletsis tubulin. The arms (inner and outer) contain dynein, which is aprotein classified as an ATPase. Dynein generates the force that resultsin a sliding movement of the microtubules, responsible for ciliarymovement. It is generally accepted that the outer dynein arms are mostlyresponsible for beating frequency whereas the inner dynein arms togetherwith the radial spokes and nexin links have a role in the waveform ofthe beating. Changes in the structural integrity of the axoneme canresult in abnormal movement that ranges from stillness to aberrantpatterns of hyperactivity.

In another aspect, provided herein is a method for delivery to ciliatedcells of a subject, comprising administrating to the subject thepharmaceutical composition as described in the present application. Insome embodiments of the method, the pharmaceutical composition comprisesa therapeutic agent (or prophylactic agent) assembled with a lipidcomposition as described in the present application, wherein the lipidcomposition comprises (i) an ionizable cationic lipid; and (ii) aselective organ targeting (SORT) lipid separate from the ionizablecationic lipid. The lipid composition may further comprise aphospholipid. In some embodiments, the ciliated cell is a lung ciliatedcell.

In some embodiments of the method, the method delivers the therapeuticagent to an organ or tissue of the subject to result in a therapeuticeffect detectable in ciliated cells in the organ or tissue of thesubject. In some embodiments, the method delivers the therapeutic agentto an organ or tissue of the subject to result in a therapeutic effectdetectable in at least about 5%, at least about 10%, at least about 15%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50% ciliated cells in the organ or tissue of the subject.

In some embodiments, the organ is lung. In some embodiments, the tissueis lung tissue. In some embodiment, the tissue is lung airway tissue. Insome embodiments, the method delivers the therapeutic agent to thesubject’s lung to result in a therapeutic effect detectable in at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, atleast about 30%, at least about 40%, at least about 50% ciliated cellsin the subject’s lung.

In some embodiments of the method, the pharmaceutical composition isadministrated to the subject through any suitable delivery. In someembodiment, the pharmaceutical composition is administrated to thesubject through inhalation. In some embodiments, the pharmaceuticalcomposition is administrated to the subject through systemicadministration such as intravenous administration.

Secretory Cell

“Secretory cell” refers to cells specialized for secretion. These cellsare usually epithelial in origin and have characteristic, well developedrough endoplasmic reticulum or, in the case of cells secreting lipids orlipid-derived products have well developed smooth endoplasmic reticulum.Examples of secretory cells include: salivary gland cells, mammary glandcells, lacrimal gland cells, creuminous gland cells, eccrine sweat glandcells, apocrine sweat gland cells, sebaceous gland cells, Bowman’s glandcells, Brunner’s gland cells, seminal vesicle cells, prostate glandcells, bulbourethral gland cells, Bartholin’s gland cells, gland ofLittre cells, endometrial cells, goblet cells of the respiratory anddigestive tracts, mucous cells of the stomach, zymogenic cells ofgastric glands, oxyntic cells of gastric glands, acinar cells of thepancreas, paneth cells of the small intestine, type II pneumocytes ofthe lung, club cells of the lung, anterior pituitary cells, cells of theintermediate pituitary, cells of the posterior pituitary, cells of thegut and respiratory tract, cells of the thyroid gland, cells of theparathyroid gland, cells of the adrenal gland, cells of the testes,cells of the ovaries, cells of the juxtaglomerular apparatus of thekidney, cells secreting extracellular matrix (e.g., epithelial cells,nonepithelial cells (such as fibroblasts, chondrocytes,osteoblasts/osteocytes, osteoprogenitor cells), and secretory cells ofthe immune system (e.g., Ig producing B cells, cytokine producing Tcells, etc.).

In another aspect, provided herein is a method for delivery to secretorycells of a subject, comprising administrating to the subject thepharmaceutical composition as described in the present application. Insome embodiments of the method, the pharmaceutical composition comprisesa therapeutic agent (or prophylactic agent) assembled with a lipidcomposition as described in the present application, wherein the lipidcomposition comprises (i) an ionizable cationic lipid; and (ii) aselective organ targeting (SORT) lipid separate from the ionizablecationic lipid. The lipid composition may further comprise aphospholipid. In some embodiments, the secretory cell is a lungsecretory cell.

In some embodiments of the method, the method delivers the therapeuticagent to an organ or tissue of the subject to result in a therapeuticeffect detectable in secretory cells in the organ or tissue of thesubject. In some embodiments, the method delivers the therapeutic agentto an organ or tissue of the subject to result in a therapeutic effectdetectable in at least about 5%, at least about 10%, at least about 15%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50% secretory cells in the organ or tissue of the subject.

In some embodiments of the method, the organ is lung. In someembodiments, the tissue is lung tissue. In some embodiment, the tissueis lung airway tissue. In some embodiments, the method delivers thetherapeutic agent to the subject’s lung to result in a therapeuticeffect detectable in at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 30%, at least about 40%,at least about 50% secretory cells in the subject’s lung.

In some embodiments of the method, the pharmaceutical composition isadministrated to the subject through any suitable delivery. In someembodiment, the pharmaceutical composition is administrated to thesubject through inhalation. In some embodiments, the pharmaceuticalcomposition is administrated to the subject through systemicadministration such as intravenous administration.

Dosing Level

In another aspect, provided is high-potency dosage form of a therapeuticagent (or prophylactic agent) formulated with a selective organtargeting (SORT) lipid, the dosage form comprising a therapeutic agent(or prophylactic agent) assembled with a lipid composition as describedherein. In some embodiments, the lipid composition comprises: (i) anionizable cationic lipid;and (ii) a selective organ targeting (SORT)lipid separate from the ionizable cationic lipid. The lipid compositionmay further comprise a phospholipid.

In some embodiments of the high-potency dosage form of the presentapplication, the SORT lipid is present in the dosage form in an amountsufficient to achieve a therapeutic effect at a dose of the therapeuticagent lower than that required with a reference lipid composition. Insome embodiments, the reference lipid composition does not comprise theSORT lipid. In some embodiments, the reference lipid composition doesnot comprise the amount of the SORT lipid. In some embodiments, thereference lipid comprises13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol(“LF92”), a phospholipid, cholesterol, and a PEG-lipid.

In some embodiments of the high-potency dosage form of the presentapplication, the SORT lipid is present in the dosage form in an amountsufficient to achieve a therapeutic effect at a dose of the therapeuticagent about 1.1-fold to about 20-fold lower than that required with areference lipid composition. In some embodiments of the high-potencydosage form of the present application, the SORT lipid is present in thedosage form in an amount sufficient to achieve a therapeutic effect at adose of the therapeutic agent about 1.1-fold to about 10-fold lower thanthat required with a reference lipid composition. In some embodiments ofthe high-potency dosage form of the present application, the SORT lipidis present in the dosage form in an amount sufficient to achieve atherapeutic effect at a dose of the therapeutic agent about 1.1-fold toabout 5-fold lower than that required with a reference lipidcomposition. In some embodiments of the high-potency dosage form of thepresent application, the SORT lipid is present in the dosage form in anamount sufficient to achieve a therapeutic effect at a dose of thetherapeutic agent about 10-fold to about 20-fold lower than thatrequired with a reference lipid composition. In some embodiments of thehigh-potency dosage form of the present application, the SORT lipid ispresent in the dosage form in an amount sufficient to achieve atherapeutic effect at a dose of the therapeutic agent at least about1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about3-fold, at least about 4-fold, at least about 5-fold, at least about6-fold, at least about 7-fold, at least about 8-fold, at least about9-fold, at least about 10-fold, at least about 11-fold, at least about12-fold, at least about 13-fold, at least about 14-fold, at least about15-fold, at least about 16-fold, at least about 17-fold, at least about18-fold, at least about 19-fold, or at least about 20-fold lower thanthat required with a reference lipid composition.

In some embodiments, the therapeutic agent is present in the dosage format a dose of about 2.0, 1.5, 1.0, 0.5, 0.2, or 0.1 milligram perkilogram (mg/kg, or mpk) body weight, or of a range between (inclusive)any two of the foregoing values.

In some embodiments, the therapeutic agent is present in the dosage format a dose of no more than about 2 milligram per kilogram (mg/kg, or mpk)body weight. In some embodiments, the therapeutic agent is present inthe dosage form at a dose of no more than about 1 milligram per kilogram(mg/kg, or mpk) body weight. In some embodiments, the therapeutic agentis present in the dosage form at a dose of no more than about 0.5milligram per kilogram (mg/kg, or mpk) body weight. In some embodiments,the therapeutic agent is present in the dosage form at a dose of no morethan about 0.2 milligram per kilogram (mg/kg, or mpk) body weight. Insome embodiments, the therapeutic agent is present in the dosage form ata dose of no more than about 0.1 milligram per kilogram (mg/kg, or mpk)body weight. In some embodiments, the therapeutic agent is present inthe dosage form at a concentration of no more than about 5 milligram permilliliter (mg/mL).

In some embodiments, the therapeutic agent is present in the dosage format a concentration of about 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 milligramper milliliter (mg/mL), or of a range between (inclusive) any two of theforegoing values.

In some embodiments, the therapeutic agent is present in the dosage format a concentration of no more than about 5 milligram per milliliter(mg/mL). In some embodiments, the therapeutic agent is present in thedosage form at a concentration of no more than about 2 milligram permilliliter (mg/mL). In some embodiments, the therapeutic agent ispresent in the dosage form at a concentration of no more than about 1milligram per milliliter (mg/mL). In some embodiments, the therapeuticagent is present in the dosage form at a concentration of no more thanabout 0.5 milligram per milliliter (mg/mL). In some embodiments, thetherapeutic agent is present in the dosage form at a concentration of nomore than about 0.1 milligram per milliliter (mg/mL).

Any suitable dosage form can be prepared for delivery, for example, viaoral, rectal, vaginal, transmucosal, pulmonary including intratrachealor inhaled, or intestinal administration; parenteral delivery, includingintramuscular, subcutaneous, intramedullary injections, as well asintrathecal, direct intraventricular, intravenous, intraperitoneal,intranasal, or intraocular injections.

In some embodiments, the dosage form can be administered in a localrather than systemic manner, for example, via injection of thepharmaceutical composition directly into a targeted tissue, preferablyin a sustained release formulation. Local delivery can be affected invarious ways, depending on the tissue to be targeted.

In some embodiments, the dosage form is an inhaled aerosol containingthe composition of the present for nasal, tracheal, or bronchialdelivery. In some embodiments, the dosage form can be provided inlozenges for oral, tracheal, or esophageal application. In someembodiments, the dosage form can be supplied in liquid, tablet orcapsule form for administration to the stomach or intestines. In someembodiments, the dosage form can be supplied in suppository form forrectal or vaginal application. In some embodiments, In some embodiments,the dosage form can be can even be delivered to the eye by use ofcreams, drops, or even injection.

In some embodiments, the administration of a dose of the therapeuticagent may be repeated.

Subject

Any subject in need thereof can be treated with the method of thepresent application. In some embodiments, the subject has beendetermined to likely respond to the therapeutic agent. For example, thesubject may have, is suffering from, or suspected of having a disease orcondition. For the example, the disease or disorder may be selected fromthe group consisting of genetic respiratory disease, chronicinflammatory lung disease, pulmonary fibrosis, central nervous system(CNS) disorder, immuno-deficiency, autoimmune disease, cancer,infectious disease, liver fibrosis, cirrhosis, metabolic disorder,muscular dystrophy, and viral infection. The therapeutic or prophylacticagent(s) as described elsewhere herein may be effective for providing atherapeutic effect for the subject by a variety of mechanisms, forexample, via gene therapy (e.g., requiring repeated administration),altered (e.g., increased) protein production, (e.g., in vivo) chimericantigen receptor (CAR) T-cell generation,, immuno-oncology,vaccine-based approach, reactivation of tumor suppressors, or othermechanisms.

In some embodiments, the subject has been determined to have a (e.g.,missense or nonsense) mutation in a target gene. In some embodiments,the mutation in the target gene is associated with a genetic disease ordisorder. In some embodiments, the target gene encodes a proteinselected from the group consisting of CFTR, DNAH5, DNAH11, BMPR2, FAH,PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9,PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A,SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, and FBN1.

In some embodiments, the subject has been determined to exhibit anaberrant expression or activity of a protein or polynucleotide thatcorresponds to a target gene. In some embodiments, the aberrantexpression or activity of the protein or polynucleotide is associatedwith a genetic disease or disorder. In some embodiments, the protein isselected from the group consisting of CFTR, DNAH5, DNAH11, BMPR2, FAH,PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9,PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A,SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, and FBN1. Insome embodiments, the polynucleotide encodes a protein selected from thegroup consisting of CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3,COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23,USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1,p53, PPT1, TPP1, hERG, PPT1, ATM, and FBN1.

In some embodiments, the subject is selected from the group consistingof mouse, rat, monkey, and human. In some embodiments, the subject is ahuman.

In another aspect, provided herein is a method for potent delivery of atherapeutic agent (or prophylactic agent) to a cell comprisingcontacting the cell with the pharmaceutical composition of the presentapplication. In some embodiments of the method, the pharmaceuticalcomposition comprises a therapeutic agent (or prophylactic agent)assembled with a lipid composition as described in the presentapplication, wherein the lipid composition comprises (i) an ionizablecationic lipid;and (iii) a selective organ targeting (SORT) lipidseparate from the ionizable cationic lipid. The lipid composition mayfurther comprise a phospholipid.

In some embodiments of the method, the cell is isolated from thesubject. In some embodiments of the method, the cell is a cell line. Insome embodiments of the method, the cell is a lung cell. In someembodiments, the lung cell is a lung airway cell. Examples of lungairway cells that can be targeted by the delivery of the presentapplication includes but is not limited to basal cell, secretory cellsuch as goblet cell and club cell, ciliated cell and any combinationthereof.

In some embodiments of the method, the SORT lipid effects a delivery ofthe therapeutic agent to the cell characterized by a greater therapeuticeffect compared to that achieved with a reference lipid composition. Insome embodiments, the reference lipid composition does not comprise theSORT lipid. In some embodiments, the reference lipid composition doesnot comprise the amount of the SORT lipid. In some embodiments, thereference lipid comprises13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol(“LF92”), a phospholipid, cholesterol, and a PEG-lipid.

In some embodiments of the method, the SORT lipid achieves about1.1-fold to about 20-fold therapeutic effect compared to that achievedwith a reference lipid composition. In some embodiments of the method,the SORT lipid achieves about 1.1-fold to about 10-fold therapeuticeffect compared to that achieved with a reference lipid composition. Insome embodiments of the method, the SORT lipid achieves about 1.1-foldto about 5-fold therapeutic effect compared to that achieved with areference lipid composition. In some embodiments of the method, the SORTlipid achieves about 10-fold to about 20-fold therapeutic effectcompared to that achieved with a reference lipid composition. In someembodiments of the method, the SORT lipid achieves at least about1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about3-fold, at least about 4-fold, at least about 5-fold, at least about6-fold, at least about 7-fold, at least about 8-fold, at least about9-fold, at least about 10-fold, at least about 11-fold, at least about12-fold, at least about 13-fold, at least about 14-fold, at least about15-fold, at least about 16-fold, at least about 17-fold, at least about18-fold, at least about 19-fold, or at least about 20-fold therapeuticeffect compared to that achieved with a reference lipid composition.

In some embodiments of the method, the SORT lipid effects delivery ofthe therapeutic agent to cells of the subject characterized by atherapeutic effect in a greater plurality of cells compared to thatachieved with a reference lipid composition. In some embodiments of themethod, the SORT lipid achieves therapeutic effect in about 1.1-fold toabout 20-fold cells compared to that achieved with a reference lipidcomposition. In some embodiments of the method, the SORT lipid achievestherapeutic effect in about 1.1-fold to about 10-fold cells compared tothat achieved with a reference lipid composition. In some embodiments ofthe method, the SORT lipid achieves therapeutic effect in about 5-foldto about 10-fold cells compared to that achieved with a reference lipidcomposition. In some embodiments of the method, the SORT lipid achievestherapeutic effect in about 10-fold to about 20-fold cells compared tothat achieved with a reference lipid composition. In some embodiments ofthe method, the SORT lipid achieves therapeutic effect in at least about1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about3-fold, at least about 4-fold, at least about 5-fold, at least about6-fold, at least about 7-fold, at least about 8-fold, at least about9-fold, at least about 10-fold, at least about 11-fold, at least about12-fold, at least about 13-fold, at least about 14-fold, at least about15-fold, at least about 16-fold, at least about 17-fold, at least about18-fold, at least about 19-fold, or at least about 20-fold cellscompared to that achieved with a reference lipid composition.

In another aspect, provided herein is a method for targeted delivery ofa therapeutic agent (or prophylactic agent) to a cell type comprisingcontacting the cell with the pharmaceutical composition of the presentapplication. In some embodiments of the method, the pharmaceuticalcomposition comprises a therapeutic agent (or prophylactic agent)assembled with a lipid composition as described in the presentapplication, wherein the lipid composition comprises (i) an ionizablecationic lipid; and (ii) a selective organ targeting (SORT) lipidseparate from the ionizable cationic lipid. The lipid composition mayfurther comprise a phospholipid.

In some embodiments of the method, the cell is isolated from thesubject. In some embodiments of the method, the cell is a cell line. Insome embodiments of the method, the cell is a lung cell. In someembodiments, the lung cell is a lung airway cell. Examples of lungairway cells that can be targeted by the delivery of the presentapplication includes but is not limited to basal cell, secretory cellsuch as goblet cell and club cell, ciliated cell and any combinationthereof.

In some embodiments of the method, the SORT lipid effects delivery ofthe therapeutic agent to cells of the subject characterized by atherapeutic effect in a first plurality of cells of a first cell typeand in a greater therapeutic effect in a second plurality of cells of asecond cell type. In some embodiments, the first cell type is differentfrom the second cell type. In some embodiments of the method, the SORTlipid achieves therapeutic effect in about 1.1-fold to about 20-foldgreater second plurality of cells of the second cell type compared tothe first plurality of cells of the first cell type. In some embodimentsof the method, the SORT lipid achieves therapeutic effect in about1.1-fold to about 10-fold greater second plurality of cells of thesecond cell type compared to the first plurality of cells of the firstcell type. In some embodiments of the method, the SORT lipid achievestherapeutic effect in about 1.1-fold to about 5-fold greater secondplurality of cells of the second cell type compared to the firstplurality of cells of the first cell type. In some embodiments of themethod, the SORT lipid achieves therapeutic effect in about 10-fold toabout 20-fold greater second plurality of cells of the second cell typecompared to the first plurality of cells of the first cell type. In someembodiments of the method, the SORT lipid achieves therapeutic effect inat least about 1.1-fold, at least about 1.5-fold, at least about 2-fold,at least about 3-fold, at least about 4-fold, at least about 5-fold, atleast about 6-fold, at least about 7-fold, at least about 8-fold, atleast about 9-fold, at least about 10-fold, at least about 11-fold, atleast about 12-fold, at least about 13-fold, at least about 14-fold, atleast about 15-fold, at least about 16-fold, at least about 17-fold, atleast about 18-fold, at least about 19-fold, or at least about 20-foldgreater second plurality of cells of the second cell type compared tothe first plurality of cells of the first cell type.

In some embodiments of the method, the SORT lipid effects delivery ofthe therapeutic agent to cells of the subject characterized by a greatertherapeutic effect in a first cell of a first cell type compared to thatin a second cell of a second cell type. In some embodiments, the secondcell type is different from the first cell type. In some embodiments ofthe method, the SORT lipid achieves about 1.1-fold to about 20-foldtherapeutic effect in first cell of the first cell type compared to thatachieved in the second cell of the second cell type. In some embodimentsof the method, the SORT lipid achieves about 1.1-fold to about 10-foldtherapeutic effect in first cell of the first cell type compared to thatachieved in the second cell of the second cell type. In some embodimentsof the method, the SORT lipid achieves about 5-fold to about 10-foldtherapeutic effect in first cell of the first cell type compared to thatachieved in the second cell of the second cell type. In some embodimentsof the method, the SORT lipid achieves about 10-fold to about 20-foldtherapeutic effect in first cell of the first cell type compared to thatachieved in the second cell of the second cell type. In some embodimentsof the method, the SORT lipid achieves at least about 1.1-fold, at leastabout 1.5-fold, at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 6-fold, at leastabout 7-fold, at least about 8-fold, at least about 9-fold, at leastabout 10-fold, at least about 11-fold, at least about 12-fold, at leastabout 13-fold, at least about 14-fold, at least about 15-fold, at leastabout 16-fold, at least about 17-fold, at least about 18-fold, at leastabout 19-fold, or at least about 20-fold therapeutic effect in firstcell of the first cell type compared to that achieved in the second cellof the second cell type.

In some embodiments of the method, the first cell type is a lung cell.In some embodiments, the first cell type is a lung airway cell. Examplesof lung airway cells that can be targeted by the delivery of the presentapplication includes but is not limited to basal cell, secretory cellsuch as goblet cell and club cell, ciliated cell and any combinationthereof.

In some embodiments of the method, the second cell type is a lung cell.In some embodiments, the second cell type is a lung airway cell.Examples of lung airway cells that can be targeted by the delivery ofthe present application includes but is not limited to basal cell,secretory cell such as goblet cell and club cell, ciliated cell and anycombination thereof.

In some embodiments, the contacting is ex vivo. In some embodiments, thecontacting is in vitro. In some embodiments, the contacting is in vivo.In some embodiments, the contacting comprises administering to a subjectthe composition comprising the therapeutic agent assembled with thelipid composition.

The following are examples of compositions and evaluations ofcompositions of the disclosure. It is understood that various otherembodiments may be practiced, given the general description providedabove.

LIST OF EMBODIMENTS

The following list of embodiments of the invention are to be consideredas disclosing various features of the invention, which features can beconsidered to be specific to the particular embodiment under which theyare discussed, or which are combinable with the various other featuresas listed in other embodiments. Thus, simply because a feature isdiscussed under one particular embodiment does not necessarily limit theuse of that feature to that embodiment.

Embodiment 1. A method for potent delivery to a lung cell of a subject,comprising: administering to said subject an aerosol compositioncomprising a therapeutic agent assembled with a lipid composition whichcomprises: (i) an ionizable cationic lipid; and (ii) a selective organtargeting (SORT) lipid separate from said ionizable cationic lipid,wherein (e.g., an amount of) said SORT lipid effects delivery of saidtherapeutic agent to said cell of said subject characterized by a (e.g.,about 1.1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-fold) greatertherapeutic effect compared to that achieved with a reference lipidcomposition (e.g., without said amount of said SORT lipid); optionally,wherein said SORT lipid is selected from those set forth in Table 6, orpharmaceutically acceptable salts thereof, or a subset of the lipids andthe pharmaceutically acceptable salts thereof.

Embodiment 2. A method for potent delivery to lung cells of a subject,comprising: administering to said subject an aerosol compositioncomprising a therapeutic agent assembled with a lipid composition whichcomprises: (i) an ionizable cationic lipid; and (ii) a selective organtargeting (SORT) lipid separate from said ionizable cationic lipid,wherein (e.g., an amount of) said SORT lipid effects delivery of saidtherapeutic agent to cells of said subject characterized by atherapeutic effect in a (e.g., about 1.1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-,9- or 10-fold) greater plurality of lung cells compared to that achievedwith a reference lipid composition (e.g., without said amount of saidSORT lipid); optionally, wherein said SORT lipid is selected from thoseset forth in Table 6, or pharmaceutically acceptable salts thereof, or asubset of the lipids and the pharmaceutically acceptable salts thereof.

Embodiment 3. A method for targeted delivery to lung cells of a subject,comprising: administering to said subject an aerosol compositioncomprising a therapeutic agent assembled with a lipid composition whichcomprises: (i) an ionizable cationic lipid; and (ii) a selective organtargeting (SORT) lipid separate from said ionizable cationic lipid,wherein (e.g., an amount of) said SORT lipid effects delivery of saidtherapeutic agent to a greater proportion of cell types as compared tothat achieved with a reference lipid composition; optionally, whereinsaid SORT lipid is selected from those set forth in Table 6, orpharmaceutically acceptable salts thereof, or a subset of the lipids andthe pharmaceutically acceptable salts thereof.

Embodiment 4. A method for targeted delivery to lung cells of a subject,comprising: administering to said subject an aerosol compositioncomprising a therapeutic agent assembled with a lipid composition whichcomprises: (i) an ionizable cationic lipid; and (ii) a selective organtargeting (SORT) lipid separate from said ionizable cationic lipid,wherein (e.g., an amount of) said SORT lipid effects delivery of saidtherapeutic agent to cells of said subject characterized by atherapeutic effect in a first plurality of lung cells of a first celltype and in a (e.g., about 1.1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or10-fold) greater second plurality of lung cells of a second cell type;optionally, wherein said SORT lipid is selected from those set forth inTable 6, or pharmaceutically acceptable salts thereof, or a subset ofthe lipids and the pharmaceutically acceptable salts thereof.

Embodiment 5. A method for targeted delivery to lung cells of a subject,comprising: administering to said subject an aerosol compositioncomprising a therapeutic agent assembled with a lipid composition whichcomprises: (i) an ionizable cationic lipid; and (ii) a selective organtargeting (SORT) lipid separate from said ionizable cationic lipid,wherein (e.g., an amount of) said SORT lipid effects a delivery of saidtherapeutic agent to cells of said subject characterized by a (e.g.,about 1.1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-fold) greatertherapeutic effect in a first lung cell of a first cell type of saidsubject compared to that in a second lung cell of a second cell type ofsaid subject, wherein said first cell type is different from said secondcell type; optionally, wherein said SORT lipid is selected from thoseset forth in Table 6, or pharmaceutically acceptable salts thereof, or asubset of the lipids and the pharmaceutically acceptable salts thereof.

Embodiment 6. The method of any one of Embodiments 1-5, wherein saidlipid composition further comprises (iii) a phospholipid.

Embodiment 7. The method of any one of Embodiments 1-6, wherein saidadministration is not intravenous administration.

Embodiment 8. The method of Embodiment 7, wherein said administrationcomprises administering by inhalation.

Embodiment 9. The method of any one of Embodiments 1-8, wherein saidlipid composition comprises said SORT lipid at a molar percentage fromabout 20% to about 65%.

Embodiment 10. The method of any one of Embodiments 1-9, wherein saidlipid composition comprises said ionizable cationic lipid at a molarpercentage from about 5% to about 30%.

Embodiment 11. The method of any one of Embodiments 1-10, wherein saidlipid composition comprises said phospholipid at a molar percentage fromabout 8% to about 23%.

Embodiment 12. The method of any one of Embodiments 1-11, wherein saidphospholipid is not an ethylphosphocholine.

Embodiment 13. The method of any one of Embodiments 1-12, wherein saidlipid composition further comprises a steroid or steroid derivative.

Embodiment 14. The method of Embodiment 13, wherein said lipidcomposition comprises said steroid or steroid derivative at a molarpercentage from about 15% to about 46%.

Embodiment 15. The method of any one of Embodiments 1-14, wherein saidlipid composition further comprises a polymer-conjugated lipid (e.g.,poly(ethylene glycol) (PEG)-conjugated lipid).

Embodiment 16. The method of Embodiment 15, wherein said lipidcomposition comprises said polymer-conjugated lipid at a molarpercentage from about 0.5% to about 10%.

Embodiment 17. The method of any one of Embodiments 1-16, wherein saidtherapeutic agent is a polynucleotide; and wherein a molar ratio ofnitrogen in said lipid composition to phosphate in said polynucleotide(N/P ratio) is no more than about 20:1.

Embodiment 18. The method of Embodiment 17, wherein said N/P ratio isfrom about 5:1 to about 20:1.

Embodiment 19. The method of any one of Embodiments 1-18, wherein amolar ratio of said therapeutic agent to total lipids of said lipidcomposition is no more than about 1:1, 1:10, 1:50, or 1:100.

Embodiment 20. The method of any one of Embodiments 1-19, wherein atleast about 85% of said therapeutic agent is encapsulated in particlesof said lipid compositions.

Embodiment 21. The method of any one of Embodiments 1-20, wherein saidlipid composition comprises a plurality of particles characterized byone or more characteristics of the following: (1) a (e.g., average) sizeof 100 nanometers (nm) or less; (2) a polydispersity index (PDI) of nomore than about 0.2; and (3) a zeta potential of -10 millivolts (mV) to10 mV.

Embodiment 22. The method of any one of Embodiments 1-21, wherein saidlipid composition has an apparent ionization constant (pKa) outside arange of 6 to 7.

Embodiment 23. The method of Embodiment 22, wherein said apparent pKa ofsaid lipid composition is of about 8 or higher.

Embodiment 24. The method of Embodiment 23, wherein said apparent pKa ofsaid lipid composition is from about 8 to about 13.

Embodiment 25. The method of any one of Embodiments 1-24, wherein saidSORT lipid comprises a permanently positively charged moiety (e.g., aquaternary ammonium ion).

Embodiment 26. The method of Embodiment 25, wherein said SORT lipidcomprises a counterion.

Embodiment 27. The method of any one of Embodiments 1-26, wherein saidSORT lipid is a phosphocholine lipid (e.g., saturated or unsaturated).

Embodiment 28. The method of any one of Embodiments 27, wherein saidSORT lipid is an ethylphosphocholine.

Embodiment 29. The method of any one of Embodiments 1-26, wherein saidSORT lipid comprises a headgroup having a structural formula:

wherein L is a (e.g., biodegradable) linker; Z⁺ is positively chargedmoiety (e.g., a quaternary ammonium ion); and X⁻is a counterion.

Embodiment 30. The method of Embodiment 29, wherein said SORT lipid hasa structural formula:

wherein R¹ and R² are each independently an optionally substitutedC₆-C₂₄ alkyl, or an optionally substituted C₆-C₂₄ alkenyl.

Embodiment 31. The method of Embodiment 29, wherein said SORT lipid hasa structural formula:

optionaly, wherein L is

wherein: p and q are each independently 1, 2, or 3; and R⁴ is anoptionally substituted C₁-C₆ alkyl.

Embodiment 32. The method of Embodiment 29, wherein said SORT lipid hasa structural formula:

wherein: R₁ and R₂ are each independently alkyl(_(C8-C24)),alkenyl(_(C8-C24)), or a substituted version of either group; R₃, R₃′,and R₃“ are each independently alkyl(_(C≤6)) or substitutedalkyl(_(C≤6)); R₄ is alkyl(_(C≤6)) or substituted alkyl(_(C≤6)); and X⁻is a monovalent anion.

Embodiment 33. The method of any one of Embodiments 1-26, wherein saidSORT lipid has a structural formula:

or

wherein: R₁ and R₂ are each independently alkyl(_(C8-C24)),alkenyl(_(C8-C24)), or a substituted version of either group; R₃, R₃′,and R₃“ are each independently alkyl(_(C≤6)) or substitutedalkyl(_(C≤6)); and X⁻ is a monovalent anion.

Embodiment 34. The method of any one of Embodiments 1-26, wherein saidSORT lipid has a structural formula:

wherein: R₄ and R₄′ are each independently alkyl(_(C6-C24)),alkenyl(_(C6-C24)), or a substituted version of either group; R₄“ isalkyl(_(C≤24)), alkenyl(_(C≤24)), or a substituted version of eithergroup; R₄”’ is alkyl(_(C1-C8)), alkenyl(_(C2-C8)), or a substitutedversion of either group; and X₂ is a monovalent anion.

Embodiment 35. The method of any one of Embodiments 1-26, wherein saidSORT lipid has a structural formula:

wherein: R₁ and R₂ are each independently alkyl(_(C8-C24)),alkenyl(_(C8-C24)), or a substituted version of either group; R₃ ishydrogen, alkyl(_(C≤6)), or substituted alkyl(_(C≤6)), or —Y1–R4,wherein: Y₁ is alkanediyl(_(C≤6)) or substituted alkanediyl(_(C≤6)); andR₄ is acyloxy(_(C≤8-24)) or substituted acyloxy(_(C≤8-24)).

Embodiment 36. The method of any one of Embodiments 1-35, wherein theionizable cationic lipid is a dendrimer or dendron of a generation (g)having a structural formula:

or a pharmaceutically acceptable salt thereof, wherein:

-   (a) the core comprises a structural formula (X_(Core)):

-   

-   wherein:    -   Q is independently at each occurrence a covalent bond, —O—, —S—,        —NR²—, or —CR^(3a)R^(3b)—_(;)    -   R² is independently at each occurrence R^(1g) or        —L²—NR^(1e)R^(1f);    -   R^(3a) and R^(3b) are each independently at each occurrence        hydrogen or an optionally substituted (e.g., C₁-C₆, such as        C₁-C₃) alkyl;    -   R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), and R^(1g) (if        present) are each independently at each occurrence a point of        connection to a branch, hydrogen, or an optionally substituted        (e.g., C₁-C₁₂) alkyl;    -   L⁰, L¹, and L² are each independently at each occurrence        selected from a covalent bond, (e.g., C₁-C₁₂, such as C₁-C₆ or        C₁-C₃) alkylene, (e.g., C₁-C₁₂, such as C₁-C₈ or C₁-C₆)        heteroalkylene (e.g., C₂-C₈ alkyleneoxide, such as        oligo(ethyleneoxide)), [(e.g., C₁-C₆) alkylene]-[(e.g., C₄-C₆)        heterocycloalkyl]-[(e.g., C₁-C₆) alkylene], [(e.g., C₁-C₆)        alkylene]-(arylene)-[(e.g., C₁-C₆) alkylene] (e.g., [(e.g.,        C₁-C₆) alkylene]-phenylene-[(e.g., C₁-C₆) alkylene]), (e.g.,        C₄-C₆) heterocycloalkyl, and arylene (e.g., phenylene); or,    -   alternatively, part of L¹ form a (e.g., C₄-C₆) heterocycloalkyl        (e.g., containing one or two nitrogen atoms and, optionally, an        additional heteroatom selected from oxygen and sulfur) with one        of R^(1c) and R^(1d); and    -   x¹ is 0, 1, 2, 3, 4, 5, or 6; and

-   (b) each branch of the plurality (N) of branches independently    comprises a structural formula (X_(Branch)):

-   

-   wherein:    -   * indicates a point of attachment of the branch to the core;    -   g is 1, 2, 3, or 4;    -   Z = 2^((g-1));    -   G=0, when g=1; or    -   $\text{G =}{\sum_{i = 0}^{i = g - 2}2^{\text{i}}},$    -   when g≠1;

-   (c) each diacyl group independently comprises a structural formula

-   

-   wherein:    -   * indicates a point of attachment of the diacyl group at the        proximal end thereof;    -   ** indicates a point of attachment of the diacyl group at the        distal end thereof;    -   Y³ is independently at each occurrence an optionally substituted        (e.g., C₁-C₁₂); alkylene, an optionally substituted (e.g.,        C₁-C₁₂) alkenylene, or an optionally substituted (e.g., C₁-C₁₂)        arenylene;    -   A¹ and A² are each independently at each occurrence —O—, —S—, or        —NR⁴—, wherein: R⁴ is hydrogen or optionally substituted (e.g.,        C₁-C₆) alkyl;    -   m¹ and m² are each independently at each occurrence 1, 2, or 3;        and    -   R^(3c), R^(3d), R^(3e), and R^(3f) are each independently at        each occurrence hydrogen or an optionally substituted (e.g.,        C₁-C₈) alkyl; and

-   (d) each linker group independently comprises a structural formula

-   

-   wherein:    -   ** indicates a point of attachment of the linker to a proximal        diacyl group;    -   *** indicates a point of attachment of the linker to a distal        diacyl group; and    -   Y₁ is independently at each occurrence an optionally substituted        (e.g., C₁-C₁₂) alkylene, an optionally substituted (e.g.,        C₁-C₁₂) alkenylene, or an optionally substituted (e.g., C₁-C₁₂)        arenylene; and

-   (e) each terminating group is independently selected from optionally    substituted (e.g., C₁-C₁₈, such as C₄-C₁₈) alkylthiol, and    optionally substituted (e.g., C₁-C₁₈, such as C₄-C₁₈) alkenylthiol.

Embodiment 37. The method of Embodiment 36, wherein x¹ is 0, 1, 2, or 3.

Embodiment 38. The method of Embodiment 36 or 37, wherein R^(1a),R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), and R^(1g) (if present) are eachindependently at each occurrence a point of connection to a branch(e.g., as indicated by *), hydrogen, or C₁-C₁₂ alkyl (e.g., C₁-C₈ alkyl,such as C₁-C₆ alkyl or C₁-C₃ alkyl), wherein the alkyl moiety isoptionally substituted with one or more substituents each independentlyselected from —OH, C₄-C₈ (e.g., C₄-C₆) heterocycloalkyl (e.g.,piperidinyl (e.g.,

), N-(C₁-C₃ alkyl)-piperidinyl (e.g.,

), piperazinyl (e.g.,

), N-(C₁-C₃ alkyl)-piperadizinyl (e.g.,

), morpholinyl (e.g.,

), N-pyrrolidinyl (e.g.,

), pyrrolidinyl (e.g.,

), or N-(C₁-C₃ alkyl)-pyrrolidinyl (e.g.,

)), and C₃-C₅ heteroaryl (e.g., imidazolyl (e.g.,

) or pyridinyl (e.g.,

)).

Embodiment 39. The method of Embodiment 38, wherein R^(1a), R^(1b),R^(1c), R^(1b), R^(1e), R^(1f), and R^(1g) (if present) are eachindependently at each occurrence a point of connection to a branch(e.g., as indicated by *), hydrogen, or C₁-C₁₂ alkyl (e.g., C₁-C₈ alkyl,such as C₁-C₆ alkyl or C₁-C₃ alkyl), wherein the alkyl moiety isoptionally substituted with one substituent —OH.

Embodiment 40. The method of any one of Embodiments 36-39, whereinR^(3a) and R^(3b) are each independently at each occurrence hydrogen.

Embodiment 41. The method of any one of Embodiments 36-40, wherein theplurality (N) of branches comprises at least 3 (e.g., at least 4, or atleast 5) branches.

Embodiment 42. The method of any one of Embodiments 36-41, wherein g=1;G=0; and Z=1.

Embodiment 43. The method of Embodiment 42, wherein each branch of theplurality of branches comprises a structural formula

Embodiment 44. The method of any one of Embodiments 36-41, wherein g=2;G=1; and Z=2.

Embodiment 45. The method of Embodiment 44, wherein each branch of theplurality of branches comprises a structural formula

Embodiment 46. The method of any one of Embodiments 36-45, wherein thecore comprises a structural formula:

(e.g.,

Embodiment 47. The method of any one of Embodiments 36-45, wherein thecore comprises a structural formula:

Embodiment 48. The method of Embodiment 47, wherein the core comprises astructural formula:

(e.g.,

Embodiment 49. The method of Embodiment 47, wherein the core comprises astructural formula:

(e.g.,

such as

Embodiment 50. The method of any one of Embodiments 36-45, wherein thecore comprises a structural formula:

wherein Q′ is —NR²— or —CR^(3a)R^(3b)—; q¹ and q² are each independently1 or 2.

Embodiment 51. The method of Embodiment 50, wherein the core comprises astructural formula:

Embodiment 52. The method of any one of Embodiments 36-45, wherein thecore comprises a structural formula

), wherein ring A is an optionally substituted aryl or an optionallysubstituted (e.g., C₃-C₁₂, such as C₃-C₅) heteroaryl.

Embodiment 53. The method of any one of Embodiments 36-45, wherein thecore comprises has a structural formula

Embodiment 54. The method of any one of Embodiments 36-45, wherein thecore comprises a structural formula selected from the group consistingof:

and pharmaceutically acceptable salts thereof, wherein * indicates apoint of attachment of the core to a branch of the plurality ofbranches.

Embodiment 55. The method of any one of Embodiments 36-45, wherein thecore comprises a structural formula selected from the group consistingof:

and pharmaceutically acceptable salts thereof, wherein * indicates apoint of attachment of the core to a branch of the plurality ofbranches.

Embodiment 56. The method of any one of Embodiments 36-45, wherein thecore comprises a structural formula selected from the group consistingof:

and pharmaceutically acceptable salts thereof, wherein * indicates apoint of attachment of the core to a branch of the plurality ofbranches.

Embodiment 57. The method of any one of Embodiments 36-45, wherein thecore has the structure

wherein * indicates a point of attachment of the core to a branch of theplurality of branches or H, wherein at least 2 (e.g., at least 3, or atleast 4) branches are attached to the core.

Embodiment 58. The method of any one of Embodiments 36-45, wherein thecore has the structure

wherein * indicates a point of attachment of the core to a branch of theplurality of branches or H, wherein at least 4 (e.g., at least 5, or atleast 6) branches are attached to the core.

Embodiment 59. The method of any one of Embodiments 36-58, wherein A¹ is—O— or —NH—.

Embodiment 60. The method of Embodiment 59, wherein A¹ is —O—.

Embodiment 61. The method of any one of Embodiments 36-60, wherein A² is—O— or —NH—.

Embodiment 62. The method of Embodiment 61, wherein A² is —O—.

Embodiment 63. The method of any one of Embodiments 36-62, wherein Y³ isC₁-C₁₂ (e.g., C₁-C₆, such as C₁-C₃) alkylene.

Embodiment 64. The method of any one of Embodiments 36-63, wherein thediacyl group independently at each occurrence comprises a structuralformula

(e.g.,

such as

), optionally wherein R^(3c), R^(3d), R^(3e), and R^(3f) are eachindependently at each occurrence hydrogen or C₁-C₃ alkyl.

Embodiment 65. The method of any one of Embodiments 36-64, wherein L⁰,L¹, and L² are each independently at each occurrence selected from acovalent bond, C₁-C₆ alkylene (e.g., C₁-C₃ alkylene), C₂-C₁₂ (e.g.,C₂-C₈) alkyleneoxide (e.g., oligo(ethyleneoxide), such as-(CH₂CH₂O)₁₋₄-(CH₂CH₂)—), [(C₁-C₄) alkylene]-[(C₄-C₆)heterocycloalkyl]-[(C₁-C₄) alkylene] (e.g.,

), and [(C₁-C₄) alkylene]-phenylene-[(C₁-C₄) alkylene] (e.g.,

Embodiment 66. The method of Embodiment 65, wherein L⁰, L¹, and L² areeach independently at each occurrence selected from C₁-C₆ alkylene(e.g., C₁-C₃ alkylene), -(C₁-C₃ alkylene-O)₁₋₄-(C₁-C₃ alkylene), -(C₁-C₃alkylene)-phenylene-(C₁-C₃ alkylene)-, and -(C₁-C₃alkylene)-piperazinyl-(C₁-C₃ alkylene)-.

Embodiment 67. The method of Embodiment 65, wherein L⁰, L¹, and L² areeach independently at each occurrence C₁-C₆ alkylene (e.g., C₁-C₃alkylene).

Embodiment 68. The method of Embodiment 65, wherein L⁰, L¹, and L² areeach independently at each occurrence C₂-C₁₂ (e.g., C₂-C₈) alkyleneoxide(e.g., -(C₁-C₃ alkylene-O)₁₋ ₄-(C₁-C₃ alkylene)).

Embodiment 69. The method of Embodiment 65, wherein L⁰, L¹, and L² areeach independently at each occurrence selected from [(C₁-C₄)alkylene]-[(C₄-C₆) heterocycloalkyl]-[(C₁-C₄) alkylene] (e.g., -(C₁-C₃alkylene)-phenylene-(C₁-C₃ alkylene)-) and [(C₁-C₄) alkylene]-[(C₄-C₆)heterocycloalkyl]-[(C₁-C₄) alkylene] (e.g., -(C₁-C₃alkylene)-piperazinyl-(C₁-C₃ alkylene)-).

Embodiment 70. The method of any one of Embodiments 36-69, wherein eachterminating group is independently C₁-C₁₈ (e.g., C₄-C₁₈) alkenylthiol orC₁-C₁₈ (e.g., C₄-C₁₈) alkylthiol, wherein the alkyl or alkenyl moiety isoptionally substituted with one or more substituents each independentlyselected from halogen, C₆-C₁₂ aryl (e.g., phenyl), C₁-C₁₂ (e.g., C₁-C₈)alkylamino (e.g., C₁-C₆ mono-alkylamino (such as -NHCH₂CH₂CH₂CH₃) orC₁-C₈ di-alkylamino (such as

)), C₄-C₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl (

), N-piperidinyl (

), N-azepanyl (

)), —OH, —C(O)OH, —C(O)N(C₁-C₃ alkyl)-(C₁-C₆ alkylene)-(C₁-C₁₂alkylamino (e.g., mono- or di-alkylamino)) (e.g.,

), -C(O)N(C₁-C₃ alkyl)-(C₁-C₆ alkylene)-(C₄-C₆ N-heterocycloalkyl)(e.g.,

), —C(O)—(C₁-C₁₂ alkylamino (e.g., mono- or di-alkylamino)), and-C(O)-(C₄-C₆ N-heterocycloalkyl) (e.g.,

), wherein the C₄-C₆ N-heterocycloalkyl moiety of any of the precedingsubstituents is optionally substituted with C₁-C₃ alkyl or C₁-C₃hydroxyalkyl.

Embodiment 71. The method of Embodiment 70, wherein each terminatinggroup is independently C₁-C₁₈ (e.g., C₄-C₁₈) alkylthiol, wherein thealkyl moiety is optionally substituted with one or more (e.g., one)substituents each independently selected from C₆-C₁₂ aryl (e.g.,phenyl), C₁-C₁₂ (e.g., C₁-C₈) alkylamino (e.g., C₁-C₆ mono-alkylamino(such as -NHCH₂CH₂CH₂CH₃) or C₁-C₈ di-alkylamino (such as

)), C₄-C₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl (

), N-piperidinyl (

), N-azepanyl (

)), —OH, —C(O)OH, —C(O)N(C₁-C₃ alkyl)-(C₁-C₆ alkylene)-(C₁-C₁₂alkylamino (e.g., mono- or di-alkylamino)) (e.g.,

), -C(O)N(C₁-C₃ alkyl)-(C₁-C₆ alkylene)-(C₄-C₆ N-heterocycloalkyl)(e.g.,

), and —C(O)—(C₄-C₆ N-heterocycloalkyl) (e.g.,

), wherein the C₄-C₆ N-heterocycloalkyl moiety of any of the precedingsubstituents is optionally substituted with C₁-C₃ alkyl or C₁-C₃hydroxyalkyl.

Embodiment 72. The method of Embodiment 71, wherein each terminatinggroup is independently C₁-C₁₈ (e.g., C₄-C₁₈) alkylthiol, wherein thealkyl moiety is optionally substituted with one substituent —OH.

Embodiment 73. The method of Embodiment 71, wherein each terminatinggroup is independently C₁-C₁₈ (e.g., C₄-C₁₈) alkylthiol, wherein thealkyl moiety is optionally substituted with one substituent selectedfrom C₁-C₁₂ (e.g., C₁-C₈) alkylamino (e.g., C₁-C₆ mono-alkylamino (suchas -NHCH₂CH₂CH₂CH₃) or C₁-C₈ di-alkylamino (such as

)) and C₄-C₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl (

), N-piperidinyl (

), N-azepanyl (

)).

Embodiment 74. The method of Embodiment 70, wherein each terminatinggroup is independently C₁-C₁₈ (e.g., C₄-C₁₈) alkenylthiol or C₁-C₁₈(e.g., C₄-C₁₈) alkylthiol.

Embodiment 75. The method of Embodiment 72 or 74, wherein eachterminating group is independently C₁-C₁₈ (e.g., C₄-C₁₈) alkylthiol.

Embodiment 76. The method of any one of Embodiments 36-69, wherein eachterminating group is independently selected from those set forth inTable 3 or a subset thereof; or wherein each terminating group isindependently selected from the group consisting of:

Embodiment 77. The method of any one of Embodiments 1-35, wherein theionizable cationic lipid is selected from those set forth in Table 4 orTable 5, or pharmaceutically acceptable salts thereof, or a subset ofthe lipids and the pharmaceutically acceptable salts thereof.

Embodiment 78. The method of any one of Embodiments 1-77, wherein saidsubject has been determined to likely respond to said therapeutic agent.

Embodiment 79. The method of any one of Embodiments 1-78, wherein saidsubject has been determined to have a (e.g., missense or nonsense)mutation in a target gene.

Embodiment 80. The method of Embodiment 79, wherein said mutation insaid target gene is associated with a genetic disease or disorder.

Embodiment 81. The method of any one of Embodiments 1-80, wherein saidsubject has been determined to exhibit an aberrant expression oractivity of a protein or polynucleotide that corresponds to a targetgene.

Embodiment 82. The method of Embodiment 81, wherein said aberrantexpression or activity of said protein or polynucleotide is associatedwith a genetic disease or disorder.

Embodiment 83. The method of any one of Embodiments 1-82, wherein saidsubject is selected from the group consisting of mouse, rat, monkey, andhuman.

Embodiment 84. The method of Embodiment 83, wherein said subject is ahuman.

Embodiment 85. The method of any one of Embodiments 1-84, wherein saidtherapeutic agent comprises a compound, a polynucleotide, a polypeptide,or a combination thereof.

Embodiment 86. The method of Embodiment 85, wherein said therapeuticagent comprises a small interfering ribonucleic acid (siRNA), a shorthairpin RNA (shRNA), a micro-ribonucleic acid (miRNA), a primarymicro-ribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), amessenger ribonucleic acid (mRNA), a clustered regularly interspacedshort palindromic repeats (CRISPR) related nucleic acid, a CRISPR-RNA(crRNA), a single guide ribonucleic acid (sgRNA), a trans-activatingCRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid(pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide(ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid,deoxyribonucleic acid (DNA), a double stranded deoxyribonucleic acid(dsDNA), a single stranded deoxyribonucleic acid (ssDNA), a singlestranded ribonucleic acid (ssRNA), a double stranded ribonucleic acid(dsRNA), a CRSIPR-associated (Cas) protein, or a combination thereof.

Embodiment 87. The method of Embodiment 86, wherein said therapeuticagent comprises a heterologous messenger ribonucleotide (mRNA); andwherein said administration results in an expression, activity, oreffect of a protein encoded by said heterologous mRNA detectable in atleast about 5%, 10%, 15%, or 20% lung epithelial cells of said subject.

Embodiment 88. The method of Embodiment 86, wherein said therapeuticagent comprises a heterologous messenger ribonucleotide (mRNA); andwherein said administration results in an expression, activity, oreffect of a protein encoded by said heterologous mRNA detectable in atleast about 2%, 5%, or 10% lung ciliated cells of said subject.

Embodiment 89. The method of Embodiment 86, wherein said therapeuticagent comprises a heterologous messenger ribonucleotide (mRNA); andwherein said administration results in an expression, activity, oreffect of a protein encoded by said heterologous mRNA detectable in atleast about 5%, 10%, 15%, or 20% lung secretory cells of said subject.

Embodiment 90. The method of Embodiment 86, wherein said therapeuticagent comprises a heterologous messenger ribonucleotide (mRNA); andwherein said administration results in an expression, activity, oreffect of a protein encoded by said heterologous mRNA detectable in atleast about 5%, 10%, 15%, or 20% lung club cells of said subject.

Embodiment 91. The method of Embodiment 86, wherein said therapeuticagent comprises a heterologous messenger ribonucleotide (mRNA); andwherein said administration results in an expression, activity, oreffect of a protein encoded by said heterologous mRNA detectable in atleast about 5%, 10%, 15%, or 20% lung goblet cells of said subject.

Embodiment 92. The method of Embodiment 86, wherein said therapeuticagent comprises a heterologous messenger ribonucleotide (mRNA); andwherein said administration results in an expression, activity, oreffect of a protein encoded by said heterologous mRNA detectable in atleast about 5%, 10%, 15%, or 20% lung basal cells of said subject.

Embodiment 93. The method of any one of Embodiments 87-92, wherein saidprotein is any one selected from the group consisting of CFTR, DNAH5,DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2,PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6,RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG,PPT1, ATM, and FBN1.

Embodiment 94. The method of any one of Embodiments 87-92, wherein saidprotein corresponds to a target gene in a lung cell (e.g., a lungepithelial cell, a lung ciliated cell, a lung secretory cell, a lungclub cell, a lung goblet cell, or a lung basal cell) of said subject.

Embodiment 95. The method of any one of Embodiment 87-92, wherein anexpression of said heterologous mRNA produces a functional variant ofsaid protein.

Embodiment 96. The method of any one of Embodiment 87-92, wherein anexpression of said heterologous mRNA increases an amount of a functionalvariant of said protein as compared to an amount of said functionalvariant of said protein generated in absence of said administration.

Embodiment 97. The method of Embodiment 86, wherein said therapeuticagent comprises a heterologous transfer ribonucleotide (tRNA) thatintroduces an amino acid into a growing peptide chain of a protein of atarget gene (e.g., at a position corresponding to a mutation in saidtarget gene encoding said protein); and wherein said administrationresults in an expression or activity of said protein detectable in atleast about 5%, 10%, 15%, or 20% lung epithelial cells of said subject.

Embodiment 98. The method of Embodiment 86 or 97, wherein saidtherapeutic agent comprises a heterologous transfer ribonucleotide(tRNA) that introduces an amino acid into a growing peptide chain of aprotein of a target gene (e.g., at a position corresponding to amutation in said target gene encoding said protein); and wherein saidadministration results in an expression or activity of said proteindetectable in at least about 2%, 5%, or 10% lung ciliated cells of saidsubject.

Embodiment 99. The method of any one of Embodiments 86 and 97-98,wherein said therapeutic agent comprises a heterologous transferribonucleotide (tRNA) that introduces an amino acid into a growingpeptide chain of a protein of a target gene (e.g., at a positioncorresponding to a mutation in said target gene encoding said protein);and wherein said administration results in an expression or activity ofsaid protein detectable in at least about 5%, 10%, 15%, or 20% lungsecretory cells of said subject.

Embodiment 100. The method of any one of Embodiments 86 and 97-99,wherein said therapeutic agent comprises a heterologous transferribonucleotide (tRNA) that introduces an amino acid into a growingpeptide chain of a protein of a target gene (e.g., at a positioncorresponding to a mutation in said target gene encoding said protein);and wherein said administration results in an expression or activity ofsaid protein detectable in at least about 5%, 10%, 15%, or 20% lung clubcells of said subject.

Embodiment 101. The method of any one of Embodiments 86 and 97-100,wherein said therapeutic agent comprises a heterologous transferribonucleotide (tRNA) that introduces an amino acid into a growingpeptide chain of a protein of a target gene (e.g., at a positioncorresponding to a mutation in said target gene encoding said protein);and wherein said administration results in an expression or activity ofsaid protein detectable in at least about 5%, 10%, 15%, or 20% lunggoblet cells of said subject.

Embodiment 102. The method of any one of Embodiments 86 and 97-101,wherein said therapeutic agent comprises a heterologous transferribonucleotide (tRNA) that introduces an amino acid into a growingpeptide chain of a protein of a target gene (e.g., at a positioncorresponding to a mutation in said target gene encoding said protein);and wherein said administration results in an expression or activity ofsaid protein detectable in at least about 5%, 10%, 15%, or 20% lungbasal cells of said subject.

Embodiment 103. The method of any one of Embodiments 97-102, whereinsaid protein is any one selected from the group consisting of CFTR,DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1,PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2,GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1,hERG, PPT1, ATM, and FBN1.

Embodiment 104. The method of any one of Embodiments 97-102, whereinsaid target gene is present in a lung cell (e.g., a lung epithelialcell, a lung ciliated cell, a lung secretory cell, a lung club cell, alung goblet cell, or a lung basal cell) of said subject.

Embodiment 105. The method of any one of Embodiments 97-102, whereinsaid tRNA reduces an amount of a non-functional variant of said proteinin said cell as compared to an amount of said non-functional variant ofsaid protein generated in absence of said contacting.

Embodiment 106. The method of Embodiment 85 or 86, wherein saidtherapeutic agent comprises a heterologous polypeptide comprising anactuator moiety, which actuator moiety is configured to complex with atarget polynucleotide corresponding to a target gene; and wherein saidadministration results in a modified expression or activity of saidtarget gene detectable in at least about 5%, 10%, 15%, or 20% lungepithelial cells of said subject, in at least about 2%, 5%, or 10% lungciliated cells of said subject, in at least about 5%, 10%, 15%, or 20%lung secretory cells of said subject, in at least about 5%, 10%, 15%, or20% lung club cells of said subject, in at least about 5%, 10%, 15%, or20% lung goblet cells of said subject, or in at least about 5%, 10%,15%, or 20% lung basal cells of said subject.

Embodiment 107. The method of Embodiment 86 or 106, wherein saidtherapeutic agent comprises a heterologous polynucleotide encoding anactuator moiety, which actuator moiety is configured to complex with atarget polynucleotide corresponding to a target gene; and wherein saidadministration results in a modified expression or activity of saidtarget gene detectable in at least about 5%, 10%, 15%, or 20% lungepithelial cells of said subject, in at least about 2%, 5%, or 10% lungciliated cells of said subject, in at least about 5%, 10%, 15%, or 20%lung secretory cells of said subject, in at least about 5%, 10%, 15%, or20% lung club cells of said subject, in at least about 5%, 10%, 15%, or20% lung goblet cells of said subject, or in at least about 5%, 10%,15%, or 20% lung basal cells of said subject.

Embodiment 108. The method of Embodiment 106 or 107, wherein saidheterologous polynucleotide encodes a guide polynucleotide configured todirect said actuator moiety to said target polynucleotide.

Embodiment 109. The method of Embodiment 106 or 107, wherein saidactuator moiety comprises a heterologous endonuclease or a fragmentthereof (e.g., directed by a guide polynucleotide to specifically bindsaid target polynucleotide).

Embodiment 110. The method of Embodiment 109, wherein said heterologousendonuclease is (1) part of a ribonucleoprotein (RNP) and (2) complexedwith said guide polynucleotide.

Embodiment 111. The method of Embodiment 109, wherein said heterologousendonuclease is part of a clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) protein complex.

Embodiment 112. The method of Embodiment 109, wherein said heterologousendonuclease is a clustered regularly interspaced short palindromicrepeats (CRISPR)-associated (Cas) endonuclease.

Embodiment 113. The method of Embodiment 109, wherein said heterologousendonuclease is selected from C2C1, C2C2, C2C3, Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas5e, Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2,Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas10, Cas10d, Cas 11, Cas12, Cas13,Cas14, CasF, CasG, CasH, CasX, CaxY, Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2,Cse3, Cse4, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16,CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or a fragment thereof.

Embodiment 114. The method of Embodiment 109, wherein said heterologousendonuclease comprises a deactivated endonuclease, optionally fused to aregulatory moiety (e.g., comprising a transcription activator, atranscription repressor, an epigenetic modifier, or a fragment thereof).

Embodiment 115. The method of Embodiment 106 or 107, wherein said targetpolynucleotide corresponds to a gene encoding any protein selected fromthe group consisting of CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA,COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A,CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9,NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, and FBN1.

Embodiment 116. The method of Embodiment 106 or 107, wherein said targetpolynucleotide corresponds to a gene in a lung cell (e.g., a lungepithelial cell, a lung ciliated cell, a lung secretory cell, a lungclub cell, a lung goblet cell, or a lung basal cell) of said subject.

Embodiment 117. The method of Embodiment 106 or 107, wherein saidexpression or activity or said modified expression or activity isdetectable at least about 4 hours after said administering.

Embodiment 118. The method of any one of Embodiments 1-117, wherein saidtherapeutic effect is characterized by an (e.g., therapeuticallyeffective) amount, activity, or effect of said therapeutic agent (e.g.,in a lung, a lung cell, a plurality of lung cells, or a lung cell typeof said subject).

Embodiment 119. The method of any one of Embodiments 1-118, wherein saidgreater therapeutic effect is characterized by a greater (e.g.,therapeutic) amount, activity, or effect of said therapeutic agent.

Embodiment 120. The method of any one of Embodiments 1-119, wherein saidreference lipid composition does not comprise said amount of said SORTlipid.

Embodiment 121. The method of Embodiment 120, wherein said referencelipid composition does not comprise said SORT lipid.

Embodiment 122. The method of any one of Embodiments 1-121, wherein saidreference lipid composition comprises13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol(“LF92”), a phospholipid, cholesterol, and a PEG-lipid.

Embodiment 123. The method of any one of Embodiments 1-122, wherein saidadministering is not intravenous administration.

Embodiment 124. The method of any one of Embodiments 1-122, wherein saidadministering comprises systemic (e.g., intravenous) administration.

Embodiment 125. The method of any one of Embodiments 1-124, wherein saidadministering comprises aerosol administration (e.g., by inhalation).

Embodiment 126. The method of any one of Embodiments 1-125, wherein saidcell comprises a lung airway cell (e.g., a lung ciliated cell, a lungsecretory cell, a lung club cell, a lung goblet cell, a lung basal cell,or a combination thereof).

Embodiment 127. The method of any one of Embodiments 1-126, wherein saidfirst cell type is ciliated cell, secretory cell, club cell, gobletcell, or basal cell.

Embodiment 128. The method of any one of Embodiments 1-127, wherein saidfirst cell type is lung (e.g., airway) cell.

Embodiment 129. The method of any one of Embodiments 1-128, wherein saidsecond cell type is ciliated cell, secretory cell, club cell, gobletcell, or basal cell.

Embodiment 130. The method of any one of Embodiments 1-129, wherein saidsecond cell type is lung (e.g., airway) cell.

Embodiment 131. An aerosol composition comprising a therapeutic agentassembled with a lipid composition, which lipid composition comprises:(i) an ionizable cationic lipid; and (ii) a selective organ targeting(SORT) lipid separate from said ionizable cationic lipid, wherein saidSORT lipid is configured to effect a delivery of said therapeutic agentcharacterized by one or more of the following: (a) a (e.g., 1.1- or10-fold) greater therapeutic effect in a lung cell of said subjectcompared to that achieved with a reference lipid composition; (b) atherapeutic effect in a (e.g., 1.1- or 10-fold) greater plurality oflung cells (e.g., of a cell type) of said subject compared to thatachieved with a reference lipid composition; (c) a therapeutic effect ina first plurality of lung cells of a first cell type and in a (e.g.,1.1- or 10-fold) greater second plurality of lung cells of a second celltype; and (d) a (e.g., 1.1- or 10-fold) greater therapeutic effect in afirst lung cell of a first cell type of said subject compared to that ina second lung cell of a second cell type of said subject, wherein theaerosol composition is formulated as an aerosol; optionally, whereinsaid SORT lipid is selected from those set forth in Table 6, orpharmaceutically acceptable salts thereof, or a subset of the lipids andthe pharmaceutically acceptable salts thereof.

Embodiment 132. The aerosol composition of Embodiment 131, wherein saidlipid composition further comprises (iii) a phospholipid.

Embodiment 133. The aerosol composition of Embodiment 131 or 132,wherein said aerosol composition comprising said therapeutic agent is apharmaceutical composition.

Embodiment 134. The aerosol composition of Embodiment 133, wherein saidaerosol droplets are generated by a nebulizer at a nebulization rate ofno more than 70 mL/minute.

Embodiment 135. The aerosol composition of Embodiment 133 or 134,wherein said aerosol droplets have a mass median aerodynamic diameter(MMAD) from about 0.5 micron (µm) to about 10 µm.

Embodiment 136. The aerosol composition of any one of Embodiments133-135, wherein said droplet size varies less than about 50% for aduration of about 24 hours under a storage condition.

Embodiment 137. The aerosol composition of any one of Embodiments133-136, wherein droplets of said aerosol composition are characterizedby a geometric standard deviation (GSD) of no more than about 3.

Embodiment 138. The aerosol composition of any one of Embodiments131-137, wherein said lipid composition comprises said SORT lipid at amolar percentage from about 20% to about 65%.

Embodiment 139. The aerosol composition of any one of Embodiments131-138, wherein said lipid composition comprises said ionizablecationic lipid at a molar percentage from about 5% to about 30%.

Embodiment 140. The aerosol composition of any one of Embodiments131-139, wherein said lipid composition comprises said phospholipid at amolar percentage from about 8% to about 23%.

Embodiment 141. The aerosol composition of any one of Embodiments131-140, wherein said phospholipid is not an ethylphosphocholine.

Embodiment 142. The aerosol composition of any one of Embodiments131-141, wherein said lipid composition further comprises a steroid orsteroid derivative.

Embodiment 143. The aerosol composition of Embodiment 142, wherein saidlipid composition comprises said steroid or steroid derivative at amolar percentage from about 15% to about 46%.

Embodiment 144. The aerosol composition of any one of Embodiments131-143, wherein said lipid composition further comprises apolymer-conjugated lipid (e.g., poly(ethylene glycol) (PEG)-conjugatedlipid).

Embodiment 145. The aerosol composition of Embodiment 144, wherein saidlipid composition comprises said polymer-conjugated lipid at a molarpercentage from about 0.5% to about 10%.

Embodiment 146. The aerosol composition of any one of Embodiments131-145, wherein said SORT lipid comprises a permanently positivelycharged moiety (e.g., a quaternary ammonium ion).

Embodiment 147. The aerosol composition of any one of Embodiments131-145, wherein said SORT lipid comprises an ionizable positivelycharged moiety (e.g., a tertiary amine).

Embodiment 148. The aerosol composition of Embodiment 146, wherein saidSORT lipid comprises a counterion.

Embodiment 149. The aerosol composition of any one of Embodiments131-148, wherein said SORT lipid is a phosphocholine lipid (e.g.,saturated or unsaturated).

Embodiment 150. The aerosol composition of any one of Embodiments 149,wherein said SORT lipid is an ethylphosphocholine.

Embodiment 151. The aerosol composition of any one of Embodiments131-150, wherein said SORT lipid comprises a headgroup having astructural formula:

wherein L is a (e.g., biodegradable) linker; Z⁺ is positively chargedmoiety (e.g., a quaternary ammonium ion); and X⁻ is a counterion.

Embodiment 152. The aerosol composition of Embodiment 151, wherein saidSORT lipid has a structural formula:

wherein R¹ and R² are each independently an optionally substitutedC₆-C₂₄ alkyl, or an optionally substituted C₆-C₂₄ alkenyl.

Embodiment 153.The aerosol composition of Embodiment 151, wherein saidSORT lipid has a structural formula:

optionaly, wherein L is

wherein: p and q are each independently 1, 2, or 3; and R⁴ is anoptionally substituted C₁-C₆ alkyl.

Embodiment 154. The aerosol composition of Embodiment 151, wherein saidSORT lipid has a structural formula:

wherein: R₁ and R₂ are each independently alkyl_((C8-C24)),alkenyl_((C8-C24)), or a substituted version of either group; R₃, R₃′,and R₃“ are each independently alkyl_((C≤6)) or substitutedalkyl(_(C≤6)); R₄ is alkyl(_(C≤6)) or substituted alkyl(_(C≤6)); and X⁻is a monovalent anion.

Embodiment 155. The aerosol composition of any one of Embodiments131-148, wherein said SORT lipid has a structural formula:

wherein: R₁ and R₂ are each independently alkyl_((C8-C24)),alkenyl(_(C8-C24)), or a substituted version of either group; R₃, R₃′,and R₃“ are each independently alkyl(_(C≤6)) or substitutedalkyl(_(C≤6)); and X⁻ is a monovalent anion.

Embodiment 156. The aerosol composition of any one of Embodiments131-148, wherein said SORT lipid has a structural formula:

wherein: R₄ and R₄′ are each independently alkyl_((C6-C24)),alkenyl_((C6-C24)), or a substituted version of either group; R₄“ isalkyl_((C≤24)), alkenyl_((C<24)), or a substituted version of eithergroup; R₄”’ is alkyl_((C1-C8)), alkenyl_((C2-C8)), or a substitutedversion of either group; and X₂ is a monovalent anion.

Embodiment 157. The aerosol composition of any one of Embodiments131-148, wherein said SORT lipid has a structural formula:

wherein: R₁ and R₂ are each independently alkyl_((C8-C24)),alkenyl(_(C8-C24)), or a substituted version of either group; R₃, R₃′,and R₃“ are each independently alkyl(_(C≤6)) or substitutedalkyl(_(C≤6)); and X⁻ is a monovalent anion.

Embodiment 158. The pharmaceutical composition of any one of Embodiments131-148, wherein said SORT lipid has a structural formula:

wherein: R₁ and R₂ are each independently alkyl_((C8-C24)),alkenyl_((C8-C24)), or a substituted version of either group; R₃ ishydrogen, alkyl_((C≤6)), or substituted alkyl_((C≤6)), or -Y₁-R₄,wherein: Y₁ is alkanediyl_((C≤6)) or substituted alkanediyl_((C≤6)); andR₄ is acyloxy_((C≤8-24)) or substituted acyloxy_((C≤8-) ₂₄₎.

Embodiment 159. The aerosol composition of any one of Embodiments131-158, wherein the ionizable cationic lipid is a dendrimer or dendronof a generation (g) having a structural formula:

or a pharmaceutically acceptable salt thereof, wherein:

-   (a) the core comprises a structural formula (X_(Core)):

-   

-   wherein:    -   Q is independently at each occurrence a covalent bond, —O—, —S—,        —NR²—, or —CR^(3a)R^(3b)—_(;)    -   R² is independently at each occurrence R^(1g) or        —L²—NR^(1e)R^(1f);    -   R^(3a) and R^(3b) are each independently at each occurrence        hydrogen or an optionally substituted (e.g., C₁-C₆, such as        C₁-C₃) alkyl;    -   R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), and R^(1g) (if        present) are each independently at each occurrence a point of        connection to a branch, hydrogen, or an optionally substituted        (e.g., C₁-C₁₂) alkyl;    -   L⁰, L¹, and L² are each independently at each occurrence        selected from a covalent bond, (e.g., C₁-C₁₂, such as C₁-C₆ or        C₁-C₃) alkylene, (e.g., C₁-C₁₂, such as C₁-C₈ or C₁-C₆)        heteroalkylene (e.g., C₂-C₈ alkyleneoxide, such as        oligo(ethyleneoxide)), [(e.g., C₁-C₆) alkylene]-[(e.g., C₄-C₆)        heterocycloalkyl]-[(e.g., C₁-C₆) alkylene], [(e.g., C₁-C₆)        alkylene]-(arylene)-[(e.g., C₁-C₆) alkylene] (e.g., [(e.g.,        C₁-C₆) alkylene]-phenylene-[(e.g., C₁-C₆) alkylene]), (e.g.,        C₄-C₆) heterocycloalkyl, and arylene (e.g., phenylene); or,    -   alternatively, part of L¹ form a (e.g., C₄-C₆) heterocycloalkyl        (e.g., containing one or two nitrogen atoms and, optionally, an        additional heteroatom selected from oxygen and sulfur) with one        of R^(1c) and R^(1d); and    -   x¹ is 0, 1, 2, 3, 4, 5, or 6; and

-   (b) each branch of the plurality (N) of branches independently    comprises a structural formula (X_(Branch)):

-   

-   wherein:    -   * indicates a point of attachment of the branch to the core;    -   g is 1, 2, 3, or 4;    -   Z = 2^((g-1));    -   G=0, when g=1; or    -   $\text{G =}{\sum_{i = 0}^{i = g - 2}2^{\text{i}}},$    -   when g≠1;

-   (c) each diacyl group independently comprises a structural formula

-   

-   wherein:    -   * indicates a point of attachment of the diacyl group at the        proximal end thereof;    -   ** indicates a point of attachment of the diacyl group at the        distal end thereof;    -   Y³ is independently at each occurrence an optionally substituted        (e.g., C₁-C₁₂); alkylene, an optionally substituted (e.g.,        C₁-C₁₂) alkenylene, or an optionally substituted (e.g., C₁-C₁₂)        arenylene;    -   A¹ and A² are each independently at each occurrence —O—, —S—, or        —NR⁴—, wherein:        -   R⁴ is hydrogen or optionally substituted (e.g., C₁-C₆)            alkyl;    -   m¹ and m² are each independently at each occurrence 1, 2, or 3;        and    -   R^(3c), R^(3d), R^(3e), and R^(3f) are each independently at        each occurrence hydrogen or an optionally substituted (e.g.,        C₁-C₈) alkyl; and

-   (d) each linker group independently comprises a structural formula

-   

-   wherein:    -   ** indicates a point of attachment of the linker to a proximal        diacyl group;    -   *** indicates a point of attachment of the linker to a distal        diacyl group; and    -   Y₁ is independently at each occurrence an optionally substituted        (e.g., C₁-C₁₂) alkylene, an optionally substituted (e.g.,        C₁-C₁₂) alkenylene, or an optionally substituted (e.g., C₁-C₁₂)        arenylene; and

-   (e) each terminating group is independently selected from optionally    substituted (e.g., C₁-C₁₈, such as C₄-C₁₈) alkylthiol, and    optionally substituted (e.g., C₁-C₁₈, such as C₄-C₁₈) alkenylthiol.

Embodiment 160. The aerosol composition of Embodiment 159, wherein x¹ is0, 1, 2, or 3.

Embodiment 161. The aerosol composition of Embodiment 159 or 160,wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), and R^(1g) (ifpresent) are each independently at each occurrence a point of connectionto a branch (e.g., as indicated by *), hydrogen, or C₁-C₁₂ alkyl (e.g.,C₁-C₈ alkyl, such as C₁-C₆ alkyl or C₁-C₃ alkyl), wherein the alkylmoiety is optionally substituted with one or more substituents eachindependently selected from —OH, C₄-C₈ (e.g., C₄-C₆) heterocycloalkyl(e.g., piperidinyl (e.g.,

), N-(C₁-C₃ alkyl)-piperidinyl (e.g.,

), piperazinyl (e.g.,

), N-(C₁-C₃ alkyl)-piperadizinyl (e.g.,

), morpholinyl (e.g.,

), N-pyrrolidinyl (e.g.,

), pyrrolidinyl (e.g.,

), or N-(C₁-C₃ alkyl)-pyrrolidinyl (e.g.,

)), and C₃-C₅ heteroaryl (e.g., imidazolyl (e.g.,

) or pyridinyl (e.g.,

)).

Embodiment 162. The aerosol composition of Embodiment 161, whereinR^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), and R^(1g) (if present)are each independently at each occurrence a point of connection to abranch (e.g., as indicated by *), hydrogen, or C₁-C₁₂ alkyl (e.g., C₁-C₈alkyl, such as C₁-C₆ alkyl or C₁-C₃ alkyl), wherein the alkyl moiety isoptionally substituted with one substituent —OH.

Embodiment 163. The aerosol composition of any one of Embodiments159-162, wherein R^(3a) and R^(3b) are each independently at eachoccurrence hydrogen.

Embodiment 164. The aerosol composition of any one of Embodiments159-163, wherein the plurality (N) of branches comprises at least 3(e.g., at least 4, or at least 5) branches.

Embodiment 165. The aerosol composition of any one of Embodiments159-164, wherein g=1; G=0; and Z=1.

Embodiment 166. The aerosol composition of Embodiment 165, wherein eachbranch of the plurality of branches comprises a structural formula

Embodiment 167. The aerosol composition of any one of Embodiments159-164, wherein g=2; G=1; and Z=2.

Embodiment 168. The aerosol composition of Embodiment 167, wherein eachbranch of the plurality of branches comprises a structural formula

Embodiment 169. The aerosol composition of any one of Embodiments159-168, wherein the core comprises a structural formula:

(e.g.,

Embodiment 170. The aerosol composition of any one of Embodiments159-168, wherein the core comprises a structural formula:

Embodiment 171. The aerosol composition of Embodiment 170, wherein thecore comprises a structural formula:

(e.g.,

Embodiment 172. The aerosol composition of Embodiment 170, wherein thecore comprises a structural formula:

(e.g.,

such as

Embodiment 173. The aerosol composition of any one of Embodiments159-168, wherein the core comprises a structural formula:

wherein Q′ is —NR²— or —CR^(3a)R^(3b)—_(;) q¹ and q² are eachindependently 1 or 2.

Embodiment 174. The aerosol composition of Embodiment 173, wherein thecore comprises a structural formula:

(e.g.,

Embodiment 175. The aerosol composition of any one of Embodiments159-168, wherein the core comprises a structural formula

(e.g.,

wherein ring A is an optionally substituted aryl or an optionallysubstituted (e.g., C₃-C₁₂, such as C₃-C₅) heteroaryl.

Embodiment 176. The aerosol composition of any one of Embodiments159-168, wherein the core comprises has a structural formula

Embodiment 177. The aerosol composition of any one of Embodiments159-168, wherein the core comprises a structural formula selected fromthe group consisting of:

and pharmaceutically acceptable salts thereof, wherein * indicates apoint of attachment of the core to a branch of the plurality ofbranches.

Embodiment 178. The aerosol composition of any one of Embodiments159-168, wherein the core comprises a structural formula selected fromthe group consisting of:

and pharmaceutically acceptable salts thereof, wherein * indicates apoint of attachment of the core to a branch of the plurality ofbranches.

Embodiment 179. The aerosol composition of any one of Embodiments159-168, wherein the core comprises a structural formula selected fromthe group consisting of:

and pharmaceutically acceptable salts thereof, wherein * indicates apoint of attachment of the core to a branch of the plurality ofbranches.

Embodiment 180. The pharmaceutical composition of any one of Embodiments159-168, wherein the core has the structure

wherein * indicates a point of attachment of the core to a branch of theplurality of branches or H, wherein at least 2 (e.g., at least 3, or atleast 4) branches are attached to the core.

Embodiment 181. The aerosol composition of any one of Embodiments159-168, wherein the core has the structure

wherein * indicates a point of attachment of the core to a branch of theplurality of branches or H, wherein at least 4 (e.g., at least 5, or atleast 6) branches are attached to the core.

Embodiment 182. The aerosol composition of any one of Embodiments159-181, wherein A¹ is —O— or —NH—.

Embodiment 183. The aerosol composition of Embodiment 182, wherein A¹ is—O—.

Embodiment 184. The aerosol composition of any one of Embodiments159-183, wherein A² is —O— or —NH—.

Embodiment 185. The aerosol composition of Embodiment 184, wherein A² is—O—.

Embodiment 186. The aerosol composition of any one of Embodiments159-185, wherein Y³ is C₁-C₁₂ (e.g., C₁-C₆, such as C₁-C₃) alkylene.

Embodiment 187. The aerosol composition of any one of Embodiments159-186, wherein the diacyl group independently at each occurrencecomprises a structural formula

(e.g.,

such as

optionally wherein R^(3c), R^(3d), R^(3e), and R^(3f) are eachindependently at each occurrence hydrogen or C₁-C₃ alkyl.

Embodiment 188. The aerosol composition of any one of Embodiments159-187, wherein L⁰, L¹, and L² are each independently at eachoccurrence selected from a covalent bond, C₁-C₆ alkylene (e.g., C₁-C₃alkylene), C₂-C₁₂ (e.g., C₂-C₈) alkyleneoxide (e.g.,oligo(ethyleneoxide), such as -(CH₂CH₂O)₁₋₄—(CH₂CH₂)—), [(C₁-C₄)alkylene]-[(C₄-C₆) heterocycloalkyl]-[(C₁-C₄) alkylene] (e.g.,

and [(C₁-C₄) alkylene]-phenylene-[(C₁-C₄) alkylene] (e.g.,

Embodiment 189. The aerosol composition of Embodiment 188, wherein L⁰,L¹, and L² are each independently at each occurrence selected from C₁-C₆alkylene (e.g., C₁-C₃ alkylene), -(C₁-C₃ alkylene-O)₁₋₄-(C₁-C₃alkylene), -(C₁-C₃ alkylene)-phenylene-(C₁-C₃ alkylene)-, and -(C₁-C₃alkylene)-piperazinyl-(C₁-C₃ alkylene)-.

Embodiment 190. The aerosol composition of Embodiment 188, wherein L⁰,L¹, and L² are each independently at each occurrence C₁-C₆ alkylene(e.g., C₁-C₃ alkylene).

Embodiment 191. The aerosol composition of Embodiment 188, wherein L⁰,L¹, and L² are each independently at each occurrence C₂-C₁₂ (e.g.,C₂-C₈) alkyleneoxide (e.g., -(C₁-C₃ alkylene-O)₁₋₄-(C₁-C₃ alkylene)).

Embodiment 192. The aerosol composition of Embodiment 188, wherein L⁰,L¹, and L² are each independently at each occurrence selected from[(C₁-C₄) alkylene]-[(C₄-C₆) heterocycloalkyl]-[(C₁-C₄) alkylene] (e.g.,-(C₁-C₃ alkylene)-phenylene-(C₁-C₃ alkylene)-) and [(C₁-C₄)alkylene]-[(C₄-C₆) heterocycloalkyl]-[(C₁-C₄) alkylene] (e.g., -(C₁-C₃alkylene)-piperazinyl-(C₁-C₃ alkylene)-).

Embodiment 193. The aerosol composition of any one of Embodiments159-192, wherein each terminating group is independently C₁-C₁₈ (e.g.,C₄-C₁₈) alkenylthiol or C₁-C₁₈ (e.g., C₄-C₁₈) alkylthiol, wherein thealkyl or alkenyl moiety is optionally substituted with one or moresubstituents each independently selected from halogen, C₆-C₁₂ aryl(e.g., phenyl), C₁-C₁₂ (e.g., C₁-C₈) alkylamino (e.g., C₁-C₆mono-alkylamino (such as -NHCH₂CH₂CH₂CH₃) or C₁-C₈ di-alkylamino (suchas

)), C₄-C₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl (

), N-piperidinyl (

), N-azepanyl (

)), —OH, —C(O)OH, —C(O)N(C₁-C₃ alkyl)-(C₁-C₆ alkylene)-(C₁-C₁₂alkylamino (e.g., mono- or di-alkylamino)) (e.g.,

), -C(O)N(C₁-C₃ alkyl)-(C₁-C₆ alkylene)-(C₄-C₆ N-heterocycloalkyl)(e.g.,

), —C(O)—(C₁-C₁₂ alkylamino (e.g., mono- or di-alkylamino)), and—C(O)—(C₄-C₆ N-heterocycloalkyl) (e.g.,

), wherein the C₄-C₆ N-heterocycloalkyl moiety of any of the precedingsubstituents is optionally substituted with C₁-C₃ alkyl or C₁-C₃hydroxyalkyl.

Embodiment 194. The aerosol composition of Embodiment 193, wherein eachterminating group is independently C₁-C₁₈ (e.g., C₄-C₁₈) alkylthiol,wherein the alkyl moiety is optionally substituted with one or more(e.g., one) substituents each independently selected from C₆-C₁₂ aryl(e.g., phenyl), C₁-C₁₂ (e.g., C₁-C₈) alkylamino (e.g., C₁-C₆mono-alkylamino (such as —NHCH₂CH₂CH₂CH₃) or C₁-C₈ di-alkylamino (suchas

)), C₄-C₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl (

), N-piperidinyl (

), N-azepanyl (

)), —OH, —C(O)OH, —C(O)N(C₁-C₃ alkyl)-(C₁-C₆ alkylene)-(C₁-C₁₂alkylamino (e.g., mono- or di-alkylamino)) (e.g.,

), -C(O)N(C₁-C₃ alkyl)-(C₁-C₆ alkylene)-(C₄-C₆ N-heterocycloalkyl)(e.g.,

), and —C(O)—(C₄-C₆ N-heterocycloalkyl) (e.g.,

), wherein the C₄-C₆ N-heterocycloalkyl moiety of any of the precedingsubstituents is optionally substituted with C₁-C₃ alkyl or C₁-C₃hydroxyalkyl.

Embodiment 195. The aerosol composition of Embodiment 194, wherein eachterminating group is independently C₁-C₁₈ (e.g., C₄-C₁₈) alkylthiol,wherein the alkyl moiety is optionally substituted with one substituent—OH.

Embodiment 196. The aerosol composition of Embodiment 194, wherein eachterminating group is independently C₁-C₁₈ (e.g., C₄-C₁₈) alkylthiol,wherein the alkyl moiety is optionally substituted with one substituentselected from C₁-C₁₂ (e.g., C₁-C₈) alkylamino (e.g., C₁-C₆mono-alkylamino (such as —NHCH₂CH₂CH₂CH₃) or C₁-C₈ di-alkylamino (suchas

)) and C₄-C₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl (

), N-piperidinyl (

), N-azepanyl (

)).

Embodiment 197. The aerosol composition of Embodiment 193, wherein eachterminating group is independently C₁-C₁₈ (e.g., C₄-C₁₈) alkenylthiol orC₁-C₁₈ (e.g., C₄-C₁₈) alkylthiol.

Embodiment 198. The aerosol composition of Embodiment 195 or 197,wherein each terminating group is independently C₁-C₁₈ (e.g., C₄-C₁₈)alkylthiol.

Embodiment 199. The aerosol composition of any one of Embodiments159-192, wherein each terminating group is independently selected fromthose set forth in Table 3 or a subset thereof; or wherein eachterminating group is independently selected from the group consistingof:

Embodiment 200. The aerosol composition of any one of Embodiments131-158, wherein the ionizable cationic lipid is selected from those setforth in Table 4 or Table 5, or pharmaceutically acceptable saltsthereof, or a subset of the lipids and the pharmaceutically acceptablesalts thereof.

Embodiment 201. A high-potency dosage form of a therapeutic agentformulated with a selective organ targeting (SORT) lipid, the dosageform comprising: said therapeutic agent assembled with a lipidcomposition that comprises: (i) an ionizable cationic lipid; and (ii)aid SORT lipid separate from said ionizable cationic lipid, wherein saidSORT lipid is present in said dosage form in an amount sufficient toachieve a therapeutic effect at a dose of said therapeutic agent (e.g.,at least about 1.1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-fold) lowerthan that required with a reference lipid composition; optionally,wherein said SORT lipid is selected from those set forth in Table 6, orpharmaceutically acceptable salts thereof, or a subset of the lipids andthe pharmaceutically acceptable salts thereof.

Embodiment 202. A high-potency dosage form of a therapeutic agentformulated with a selective organ targeting (SORT) lipid, the dosageform comprising: said therapeutic agent assembled with a lipidcomposition that comprises: (i) an ionizable cationic lipid; and (ii)said SORT lipid separate from said ionizable cationic lipid, whereinsaid therapeutic agent (e.g., heterologous polynucleotide) is present insaid dosage form at a dose of no more than about 2 milligram perkilogram (mg/kg, or mpk) body weight; optionally, wherein said SORTlipid is selected from those set forth in Table 6, or pharmaceuticallyacceptable salts thereof, or a subset of the lipids and thepharmaceutically acceptable salts thereof.

Embodiment 203. The dosage form of Embodiment 201 or 202, wherein saidlipid composition further comprises (iii) a phospholipid.

Embodiment 204. The dosage form of any one of Embodiments 201 -203,wherein said dosage form is an aerosol dosage form.

Embodiment 205. The dosage form of any one of Embodiments 201- 203,wherein said dosage form is an intravenous dosage form.

Embodiment 206. The dosage form of any one of Embodiments 201-205,wherein said dosage form is for lung delivery.

Embodiment 207. The dosage form of any one of Embodiments 201-206,wherein said therapeutic agent (e.g., heterologous polynucleotide) ispresent in said dosage form at a dose of no more than about 1.0, 0.5,0.1, 0.05, or 0.01 mg/kg body weight.

Embodiment 208. The dosage form of any one of Embodiments 201-207,wherein said therapeutic agent (e.g., heterologous polynucleotide) ispresent in said dosage form at a concentration of no more than about 5or 2 milligram per milliliter (mg/mL).

Embodiment 209. The dosage form of any one of Embodiments 201-208,wherein the dosage form is formulated as an aerosol compositionaccording to any one of Embodiments 131-200.

Embodiment 210. A method for delivery by nebulization to lung cell(s) ofa subject, the method comprising: administering to said subject anaerosol composition comprising a therapeutic agent assembled with alipid composition, which lipid composition comprises: (i) an ionizablecationic lipid; and (ii) a selective organ targeting (SORT) lipidseparate from said ionizable cationic lipid, thereby delivering saidtherapeutic agent to said lung cell(s) of a lung of said subject;optionally, wherein said SORT lipid is selected from those set forth inTable 6, or pharmaceutically acceptable salts thereof, or a subset ofthe lipids and the pharmaceutically acceptable salts thereof.

Embodiment 211. The method of Embodiment 210, wherein the methodprovides a (e.g., therapeutically) effective amount or activity of saidtherapeutic agent in at least about 5%, 10%, 15%, or 20% lung epithelialcells of said subject.

Embodiment 212. The method of Embodiment 210 or 211, wherein the methodprovides a (e.g., therapeutically) effective amount or activity of saidtherapeutic agent in at least about 2%, 5%, or 10% lung ciliated cellsof said subject.

Embodiment 213. The method of any one of Embodiments 210-212, whereinthe method provides a (e.g., therapeutically) effective amount oractivity of said therapeutic agent in at least about 5%, 10%, 15%, or20% lung secretory cells of said subject.

Embodiment 214. The method of any one of Embodiments 210-213, whereinthe method provides a (e.g., therapeutically) effective amount oractivity of said therapeutic agent in at least about 5%, 10%, 15%, or20% lung club cells of said subject.

Embodiment 215. The method of any one of Embodiments 210-214, whereinthe method provides a (e.g., therapeutically) effective amount oractivity of said therapeutic agent in at least about 5%, 10%, 15%, or20% lung goblet cells of said subject.

Embodiment 216. The method of any one of Embodiments 210-215, whereinthe method provides a (e.g., therapeutically) effective amount oractivity of said therapeutic agent in at least about 5%, 10%, 15%, or20% lung basal cells of said subject.

Embodiment 217. The method of any one of Embodiments 210-216, whereinsaid lipid composition comprises a phospholipid.

Embodiment 218. The method of any one of Embodiments 210-217, whereinsaid aerosol composition comprising said therapeutic agent assembledwith said lipid composition is an aerosol composition.

Embodiment 219. The method of any one of Embodiments 210-218, whereinthe aerosol composition is formulated according to any one ofEmbodiments 131-200.

EXAMPLES Example 1. Preparation of DOTAP or DODAP Modified LipidNanoparticles

Lipid nanoparticles (LNPs) are the most efficacious carrier class for invivo nucleic acid delivery. Historically, effective LNPs are composed of4 components: an ionizable cationic lipid, zwitterionic phospholipid,cholesterol, and lipid poly(ethylene glycol) (PEG). However, these LNPsresult in only general delivery of nucleic acids, rather than organ ortissue targeted delivery. LNPs typically delivery RNAs only to theliver. Therefore, new formulations of LNPs were sought in an effort toprovide targeted nucleic acid delivery.

The four canonical types of lipids were mixed in a 15:15:30:3 molarratio, with or without the addition of a permanently cationic lipid.Briefly, LNPs were prepared by mixing a dendrimer or dendron lipid(ionizable cationic), DOPE (zwitterionic), cholesterol, DMG-PEG, andDOTAP (permanently cationic). Alternatively DOTAP can be substituted forDODAP to generate a LNP comprising DODAP. The structure of DODAP andDODAP are shown in FIG. 1 . Various dendrimer or dendron lipids that maybe used are shown in FIG. 2 .

For preparation of the LNP formulation, a dendrimer or dendron lipid,DOPE, Cholesterol and DMG-PEG were dissolved in ethanol at desired molarratios. The mRNA was dissolved in citrate buffer (10 mM, pH 4.0). ThemRNA was then diluted into the lipids solution to achieve a weight ratioof 40:1 (total lipids:mRNA) by rapidly mixing the mRNA into the lipidssolution at a volume ratio of 3:1 (mRNA:lipids, v/v). This solution wasthen incubated for 10 min at room temperature. For formation of DOTAPmodified LNP formulations, mRNA was dissolved in 1 × PBS or citratebuffer (10 mM, pH 4.0), and mixed rapidly into ethanol containing5A2-SC8, DOPE, Cholesterol, DMG-PEG and DOTAP, fixing the weight ratioof 40:1 (total lipids:mRNA) and volume ratio of 3:1 (mRNA:lipids).Formulation are named X% DOTAP Y (or X%DODAP Y) where X represents theDOTAP (or DODAP) molar percentage in total lipids, and Y represents thetype of dendrimer or dendron lipid. Alternatively, formulation may benamed Y X%DOTAP or Y X%DODAP where X represents the DOTAP (or DODAP)molar percentage in total lipids, and Y represents the type of dendrimeror dendron lipid.

Example 2. SORT LNP Stability

LNPs were tested for stability. 5A2-SC8 20% DODAP (“Liver-SORT) and5A2-SC8 50% DOTAP (“Lung-SORT”) were generated using either amicrofluidic mixing method or a cross/tee mixing method. The differentLNP formulations were characterized by size, polydispersity index (PDI)and zeta-potential, were examined by dynamic light scattering, 3separate times for each formulation. The characteristics of the LNPs areshow in Table 8.

TABLE 8 SORT LNP characteristics Size (nm) PDI Zeta (mV) EncapsulationEfficiency (%) Lung-SORT -microfluidic 82.3 0.10 3.0 100 Lung-SORT-cross/tee mixing 78.1 0.09 2.2 100 Liver-SORT -microfluidic 59.1 0.10-2.3 97 Liver-SORT -cross/tee mixing 60.0 0.11 -30 96

The encapsulation efficiency was tested using a Ribogreen RNA assay(Zhao et al., 2016). Briefly, mRNA was encapsulated with > 95%efficiency in LNPs when the mRNA was dissolved in acidic buffer (10 mMcitrate, pH 4). The characteristics were observed over 28 days for thetwo types of LNPs (5A2-SC8 20% DODAP (“Liver-SORT) and 5A2-SC8 50% DOTAP(“Lung-SORT”)). FIG. 6 shows the changes of the characteristics of theLNP over the course of 28 days.

In addition, to the measure of the stability of the LNPs in solution,the stability of the LNPs and resulting mRNA expression was observed inmice. Briefly, mice were injected intravenously with 0.1 mg/kg andobserved in vivo. Luciferin was added 5 hrs. after injection andvisualized. As shown in FIG. 7 , the Lung-SORT LNP generated tissuespecific radiance in the lungs which remained high even after 14 daywith a slight decay in signal by the 21^(st) and 28^(th) day. FIG. 8shows images of the organs of the mouse at specific times periods aftertreated with Lung-SORT or Liver-SORT.

Example 3. Expression of TR mRNA in Different Cell Types

TR mRNA was loaded into either 20%DODAP 4A3-SC7 LNP or 10%DOTAP 5A2-SC8LNPs and delivered into well-differentiated human bronchial epithelialcultures using apical bolus dosing. Cell expression was observed invarious cell type and the percent of the cell type that expressed TR wasplotted. As shown in the top panel of FIG. 3 , the 20% DODAP 4A3-SC7LNPs preferentially caused secretory cells to express TR, while 10%DOTAP5A2-SC8 LNPs cause the ciliated cells to preferentially express TR. Thispreferential delivery may allow a treatment delivered to the lungs topreferentially affect a specific cell type in the lungs The TR mRNA wasalso loaded into LNPs without the SORT lipid (e.g. DODAP or DOTAP) toidentify how the DODAP or DOTAP affected the potency. As shown in thebottom panel of FIG. 5 , the LNPs comprising DOTAP or DODAP showedincrease TR expression compared to their corresponding LNP without DOTAPor DODAP.

Example 4. Luciferase Activity and Histopathology From LNPs Deliveredvia Inhaled Aerosol

Luc mRNA was loaded into a number of LNPs including LNPs of comprising aSORT lipid and a dendrimer or dendron. LNPs of 4A3-SC7 20% DODAP,4A3-SC7 10% DODAP, 5A2-SC8, 5A2-SC8 10% DOTAP were generated and loadedwith Luc mRNA. 0.4/2/8 mg of LNP-formulatedLuc2 mRNA (1 mg/ml) wasdelivered into the pie chamber by nebulization (Aerogen solo), with anestimated (not measured) per mouse delivered dose of 0.01, 0.06 or 0.22mg/kg. The mice were 7-week-old B6 male albino mice. Luciferin wasadministered to the mice 5 hrs. after delivery of the LNPs. Theluciferase activity was detected as a measure of delivery to a target.FIG. 4 shows the distribution and expression of the luciferase in themice demonstrating the expression was successful and delivery of theLNPs could be performed using inhaled aerosol delivery.

Example 5. Toxicity of EPC Containing LNPs

LNPs comprising ethylphosphocholine (EPC) in place of DOTAP or DODAPwere tested for toxicity by using apical bolus dosing on human bronchialepithelial cells. The % of lactate dehydrogenase (LDH) that was releasedwas used as a metric of cellular death and indicative of the toxicity ofthe LNP. The release of LDH was detected prior to treatment(pretreatment) and 24 post treatment. As shown in FIG. 5 , the treatmentof 50% DOTAP LNP resulted in an ~15% LDH release whereas EPC didn’t showa significant %LDH release. Importantly, DOTAP and EPC have a similarquaternary amine moiety, indicating that the activity for cell targetingmay be similar, but that EPC is considerably less toxic.

Example 6. Production of DNAI1 mRNA

DNA corresponding to the gene of DNAI1 was synthesized at GenScript.pUC57/DNAI1 was digested with HindIII and EcoRI HF restriction enzymes.Moreover, a digested pVAX120 vector and DNAI1 cDNA were gel purified andligated (the ORF for DNAI1 is codon optimized). Standard in vitrotranslation procedure was used for RNA production utilizing unmodifiednucleotides. Capping reaction was carried out using Vaccinia Viruscapping system and cap 2′—O—methyl transferase.

Example 7 Detection of DNAI1 mRNA Delivery to a Subject

A subject having or suspected of having primary ciliary dyskinesia (PCD)is given a treatment by administering a composition as describedelsewhere herein. The subject is monitored at regular intervals forexpression of DNAI1 in the lungs. A sample of lung tissue from thesubject is taken comprising ciliated cells of the lung. The cells areharvested and prepared for RNA isolation. cDNA is produced from the RNAusing a first strand synthesis kit and random hexamer. qPCR reactionsare run using a set of forward and reverse primers and a fluorescentprobe, specific to DNAI1 and a second set specific to a control orhousekeeping gene for expression normalization. Expression of DNAI1 isdetected using a fluorescent readout corresponding the DNAI1 probe.

Example 8. Functional Rescue in hBEs With DNAI1 mRNA

Repeated doses of lipid compositions described herein were delivered tohuman basal epithelial cells (hBEs) deficient in DNAI1 as described inthe first column of FIG. 10A. Results of the study are summarized in theleft column of FIG. 10E. Cellular uptake was observed in the presence ofmucus. The activity of cilia post treatment was comparable to normalcontrols. Normal beat frequency and synchronized wave-like motion ofcilia was recovered. The first column of FIG. 10B further illustratestargeting of DNAI1-HA mRNA to ciliated cells, Immunofluorescence ofDNAI1-HA and acetylated tubulin (biomarker for ciliated cells) shows theexpression of DNAI1-HA in hBEs 72 h after dosing.

Example 9. Potency and Tolerability Study in Mice

Single administration and escalating doses of lipid compositionsdescribed herein comprising a luciferase mRNA payload are tested forpotency and tolerability in mice. Key features of the study aresummarized in the middle column of FIG. 10A. Mice are treated with alipid composition comprising an ionizable cationic lipid (e.g., 4A3SC7,5A2SC8) and a SORT lipid (e.g., DODAP, DOTAP) aerosolized via anebulizer. Luciferase expression is measured to assess potency andhistopathology is measured to asses tolerability. Good distribution andhigh levels of protein expression are observed in whole-body images ofmice treated with the lipid composition, such as that depicted in themiddle column of FIG. 10E. Histopathology results are comparable tocontrol animals indicating high tolerability. The results from testing ashort delivery time (e.g., 5-8 min) and low concentration used (e.g.,0.5 mg/mL) provides support for increasing the dosage.

Example 10. DNAI1 Expression in Lung Tissues of NHPs

Two lipid compositions, RTX0001 (5 components) and RTX0004 (4components), were evaluated in a non-human primate (NHP, cynomolgusmacaques) study to demonstrate DNAI1 expression in lung tissues. RTX0001comprises 4A3-SC7, DODAP, DOPE, cholesterol, and DMG-PEG at a molarratio of 19.05:20:19.05:38.09:3.81, respectfully. RTX0004 comprises5A2-SC8, DOPE, cholesterol, and DMG-PEG at a molar ration of19.05:23.81:47.62:4.76, respectfully. Key features of the study aresummarized in the fourth column from the left of FIG. 10A. Furtherexperimental details are summarized in the middle column of FIG. 10B.Briefly, two formulations, one comprising a SORT LNP formulation(comprising, e.g., DODAP, DOTAP) and another comprising an LNPformulation were delivered to NHPs as a single dose via intubation. Bothcompositions contained DNAI1-HA mRNA. DNAI-HA mRNA and DNAI1-HA proteinexpression were detected in lungs of NHPs at 6 and 24 hours at doses ofthe aerosolized compositions of 0.1 mg/kg or less. The right column ofFIG. 10C shows DNAI1-HA mRNA and corresponding protein expressionobserved 6 hours post treatment in the lung tissues of treated NHPs. Thecomposition comprising a SORT molecule resulted in stronger observedexpression of DNAI1-HA and DNAI1-HA mRNA in the lungs of NHPs. Noadverse clinical observation or tolerability issues were detectedprecluding use of the formulations at higher doses or in multi-dosesettings. FIG. 10D shows that that by replacing 100% of U’s in the mRNAwith modified nucleotide m1Ψ minimized cytokine response.

Example 11. Screening Lipid Formulations

Lipid compositions comprising an ionizable cationic lipid (e.g., 4A3SC7,5A2SC8) with or without a SORT lipid (e.g., DODAP, DOTAP) were screenedto enable longer storage and shipping, decrease required dose, shortennebulization times (e.g., nebulization flow rate), and increasetolerability, as described in the second column from the left of FIG.10A. Lipid compositions were screened by changing the ionizable lipid,the SORT lipid, buffer identity (e.g., PBS) and concentration, saltidentity (e.g., NaCl) and concentration, cryopreservative identity(e.g., sucrose, trehalose, mannitol, xylitol, lactose) andconcentration, N/P ratio, and PEG content. For screening N/P ratios: theFA, psd, %free, and yield were recorded, the potency andtolerability/toxicity (e.g., ciliary activity, LDH, cytokines) areassessed, and the nebulization time were evaluated (e.g., single dose,mice model). Various formulations were evaluated on the basis ofparticle size, polydispersity index (PDI), encapsulation efficiency(percent free mRNA). The lipid compositions were then screened forpotency, targeting efficiency/specificity, stability, andtolerability/toxicity (e.g., ciliary activity, LDH, cytokines, bloodchemistry markers) in human basal epithelial (hBE) cell cultures, mousemodels, and mouse models for NHPs.

Experimental details and readouts are summarized in the second columnfrom the left of FIG. 10A. Three different formulations with varying N/Pratios are summarized in Table 9.

TABLE 9 Lipid formulations tested for potency, stability, andtolerability Formulation A Formulation B Formulation CConcentration/potency 0.5 mg/mL 1 mg/mL 1-2 mg/mL Human dose Est ≥ 0.5mg/kg 0.1-0.5 mg/kg 0.1-0.3 mg/kg Administration Frequency TBD Once ortwice per week Once or twice per two weeks Tolerability TBD Adequate tosupport administration frequency Adequate to support administrationfrequency Sterilization Sterile filtration Sterile filtration Sterilefiltration Stability / shelf life 2-4 weeks at 2-8° C. (cannot befrozen) ≥ 1 year at -80° C.; after thaw at 5° C. ≥ 1 week ≥ 2 year at-20° C.; after thaw 5° C. 1 month Alternatively, lyophilized, stored at2-8° C. ≥ 2 years Nebulization: Flow rate > 0.2 mL/min at 0.5 mg/mL 0.4mL/min at 0.5 mg/mL > 0.4 mL/min at 0.5 mg/mL Device Nebulization timeAerogen solo TBD Aerogen Solo or Pari 30-60 min Breath-actuated 30 minor less

Moreover, aerosol compositions without the presence of lipidcompositions (e.g., only salt(s), buffer(s), cryopreservative(s)) aretested to determine effect of each component and its concentration onthe nebulization flow rate.

The aerosolized lipid compositions may provide synergy with theadditional administration of the lipid composition through IV.

Example 12. Clinical Study of mRNA Treatment for Primary CiliaryDyskinesia

Adult subjects having or suspected of having primary ciliary dyskinesia(PCD) are given a treatment by administering a composition as describedelsewhere herein. Subjects may be selected on the basis of a pathogenicmutation in DNAI1 and/or FEV1 between 40% and 90%. The study is singleascending dose (SAD), multiple ascending dose (MAD), or open-labelextension (OLE) study. Subjects are sorted into placebo, low dose, andhigh dose treatment groups and receive a corresponding amount offormulation (or placebo). Subjects are observed for safety andtolerability and absolute change in percent predicted FEV1. Subjectstreated with the formulation show signs of high tolerability andincrease in percent predicted FEV1. FIG. 9A summarizes the maincomponents of such a study. FIG. 9B illustrates an ex vivo model ofciliated epithelial cells (mouse tracheal epithelial cells or MTECscultured at an air-liquid Interface (ALI) for testing the efficacy ofrescue by the DNAI1 mRNA treatment described herein. MTECs were obtainedfrom PCD conditional KO mouse model (Dnaic1 KO) and cultured at anair-liquid interface. The cells (ciliated, goblet, or basal) formedtight junctions and produced mucus, thus remodeling and restoringproperties similar the native epithelium. FIG. 9C illustrates thatciliary activity in KO mouse cells was rescued by the DNAI1 mRNAtreatment, and the treatment effect remained stable for weeks afterdosing was stopped. Dnaic 1 KO mouse cells were treated basally threetimes a week starting on day 7 during differentiation. The last dose wasArmentieres on day 19. Ciliary activity in treated Dnaic1 KO culture wasfirst detected 5 days after dosing was initiated. Activity in treatedDnaic1 KO cells reached 36% of normal (vs PCD/no TAM controls) cells byday 24. Ciliary activity in treated Dnaic1 KO cells remained above 20%of normal (more than 50% of max) cells 23 days after the last treatment(the last timepoint assessed). No ciliary activity was detected inuntreated Dnaic1 KO cultures throughout the duration of the study.

Example 13. Dose Finding and Repeat Administration Studies in NHPs

Lipid compositions as described herein are used in dose finding andrepeated administration studies in non-human primates (NHPs). Anoverview of such a study is described in the rightmost column of FIG.10A. Lipid compositions are tested to determine clinical candidates foran investigational new drug study, determine appropriate dose and dosingfrequency, determine the maximum tolerated dose, and select anebulization device for clinical development. The three rightmostcolumns FIG. 10B summarize these and other goals of the study.Experimental readouts are pharmacokinetics (PK), tolerability,biodistribution, and immunological response as measured by techniquesdescribed above.

Example 14. Detection of DNAI1-HA mRNA Expression in Cells

Human DNAI1 knock-down cells were cultured at an air-liquid interface(ALI). The cultured cells were treated with a single dose of LNPscontaining DNAI1-HA mRNA (10 µg/mL of media). Cells were immunostainedwith anti-acetylated tubulin (ciliated cell marker) and anti-HAantibodies 24 hours, 48 hours, 7 days, and 14 days after dosing. FIG.11A shows immunofluorescence imaging of these cells at the indicatedtimepoints, demonstrating targeted cells successfully expressed theDNAI1-HA mRNA. Integration of DNAI1-HA into axoneme of cilia is seen topeak between 48-72 hours after treatment. Well-differentiated humanDNAI1 knock-down cells were treated with a single dose of a formulationof DNAI1-HA mRNA described herein and immunostained with anti-acetylatedtubulin and anti-HA. Integration of newly-expressed DNAI1-HA intoaxonmeme of cilia peaked between 48 to 72 hours after treatment.DNAI1-HA was detected in ciliary axoneme for more than 24 days aftersingle administration. Repeated administration resulted in rescue ofciliary activity that remained for weeks after the dosing was stopped.FIG. 11B illustrates that newly-made HA-tagged DNAI1 was rapidlyincorporated into the cilia of human bronchial epithelial cells (hBEs).Well-differentiated human DNAI1 knock-down cells were treated (basaladministration) with a single dose of LNP formulated DNAI1-HA (10 µg in2 ml of media). Cells were immunostained with anti-acetylated tubulinand anti-HA 72 hours after dosing. More than 90% of ciliated cells waspositive for DNAI1-HA.

Example 15. Biomarkers and Multiplex Immunofluorescence Panel forEpithelial Cell Types

A multiplexed immuncofluroescence panel was developed to distinguishcertain epithelial cell types on the basis of certain biomarkers. Theparticular biomarkers and corresponding cell types targeted in the panelare summarized in Table 10. FIG. 12 shows the results of the panel. Ineach panel, the corresponding cell type is identified viaimmunofluorescence by the presence of a biomarker or biomarkers.

TABLE 10 Multiplex IF Panel cell types and corresponding markers CellType Example Marker Epithelium EPCAM Ciliated acetylated-tubulin(AC-Tubulin) Club Secretoglobin Family 1A Member 1 (SCGB1A1) GobletMucin 5AC (MUC5AC) Basal (stem) Cytokeratin 5 (CK5)

Example 16. Observation of Specific Cell Tropism Signatures in LNPFormulations

Well-differentiated human bronchial epithelial (hBE) cells were treatedonce with either LNP A, B, or D (200 µg) using Vitrocell nebulization.Ciliated cells, basal cells, club cells, and goblet cells weredistinguished on the basis of cell markers as detailed in Table 11. FIG.13A shows the percentage of each cell type successfully transfected withtdTomato mRNA as measured by percentage of each cell type expressingtdTomato (% TR positive) and demonstrates specific cell tropismsignatures for each LNP formulation. FIG. 13B shows illustrates aerosoladministration of formulated DNAI1 mRNA rescued ciliary activity inknock-down primary hBE ALI cultures. Well-differentiated human DNAI1-knock-down cells (hBEs) were treated 2 times per week withLNP-formulated DNAi1 (300 µg per Vitrocell nebulization) starting on day25 post ALI (culture age). Last dose was administered on day 50 postALI. Increased ciliary activity in treated DNAI1 knock-down cultures wasfirst detected seven days after dosing was initiated. Rescued ciliaryactivity had normal beat frequency (9-17 Hz) and appeared synchronized.

TABLE 11 Cell markers for distinguishing cell types Ciliated cellacylated-tubulin (Ac-Tubulin) Basal cell Cytokeratin 5 (CK5) Club cellSecretoglobin Family 1A Member 1 (SCGB1A1) Goblet cell Mucin 5AC(MUC5AC)

Example 17. Expression of Tomato Red (TR) in Basal and Secretory Cells

Expression of TR (Tomato Red) mRNA in different cell types in HBEcultures (human bronchial epithelial cultures) was analyzed. TR mRNA wasloaded into one of 20% DODAP-4A3 SC7 40:⅟PBS (FIG. 14A,), 4A3-SC7-20%DODAP 40:⅟Buffer 27/frozen (FIG. 14B), 4A3-SC7-20% DODAP 30:⅟Buffer27/frozen (FIG. 14C), 4A3-SC7 20% 14:0 EPC, 30:1 /Buffer 27/frozen (FIG.14D), 4A3-SC7 20% 14:0 TAP, 30:1 /Buffer 27, frozen (FIG. 14E) anddelivered into well-differentiated human bronchial epithelial culturesby aerosol delivery. TR protein expression in various cell-types wasobserved and the percent positive TR cells in different cell-types wasplotted. Cell types observed and corresponding cell markers are asdescribed in Example 16. As shown in each panel of FIG. 14 , TR was seenprimarily in basal and secretory cells in treated cultures. Note thatHBE cultures have high numbers of goblet cells.

Example 18. DNAI1-HA Is Expressed in Cells of the Respiratory EpitheliumFrom NHP Lung Samples

Non-human primates (NHPs) were treated by aerosol delivery with a lowdose of DNAI1-HA mRNA contained in lipid compositions comprising a SORTlipid as described herein. Multiplex immunofluorescence was used toquantify DNAI1-HA expression in lung tissue blocks from treated animals.Lung tissue blocks were analyzed 6 h after treatment (6 hrs) or 24 hafter treatment (24 hrs) with lipid compositions containing buffer as acontrol (vehicle). Cell markers for each cell type are as detailed inExample 6. As shown in FIG. 15 and FIGS. 16A-B, DNAI1-HA expression wasdetected in lung samples from NHPs treated with the lipid compositioncontaining DNAI1-HA mRNA. Further, DNAI1-HA expression co-localized withmarkers for epithelial cells, including club, basal, and ciliated cells,indicating the lipid composition preferentially targeted the respiratoryepithelium. Single 0.4 mg/kg administration of inhaled LNP-formulatedDNAI1-HA mRNA was introduced to two NHPs (one male and one female). Lungand bronchial sections were collected six hours after dosing. Percentageof DNAI1-HA positive was calculated by combining cell counts from 4 lungsections (~500,000 to 1,400,0000 cells counted) and 1 bronchial section(~16,000 to 65,000 cells counted) from each animal.

Example 19. Aerosol Administration of Formulated DNAI1 mRNA RescuesCiliary Activity in Knock-Down Primary Bronchial Human ALI Cultures

Human primary bronchial epithelial DNAI1 knock-down cells were culturedat an ALI. Well-differentiated cells were treated 2x/week (T, F) withLNP C (300 µg/d using Vitrocell nebulization) starting on day 25 postALI culture age. The last does was administered on day 50 post ALIculture. Ciliary activity was measured by cross-sectional area (CSA) andbeat frequency following certain doses. FIG. 17 shows increased ciliaryactivity in treated DNAI1 knock-down cultures was first detected 7 daysafter dosing was initiated.

Example 20. Prolonged Rescue of Ciliary Activity in KO-Primary TrachealMouse ALI Cultures

Mouse tracheal epithelial cells (MTEC) are harvested from Dnaic1 mice.The cells are cultured as described in Example 8 and grown untildifferentiating. The differentiating Dnaic 1 knock-out (KO) mouse cellsare treated with a low dose of a lipid composition comprising a SORTcompound disclosed herein and carrying a DNAI1 mRNA. Ciliary activity asmeasured by ciliary cross-sectional area (CSA) and ciliary beatfrequency (CSF) is determined at certain timepoints. Wild type (WT) andPCD/no TAM cells are used as positive controls and untreated Dnaic1 KOcells as negative controls. Ciliary activity in treated Dnaic1 KO cellsmay be higher than untreated Dnaic1 KO cells.

Example 21. Additional SORT Molecules

SORT lipids were screened for strong lung or spleen specificity andtolerability in mouse IV studies. The second from the left column ofFIG. 10B discusses the cell tropism achieved through the SORT lipidsscreened. Five SORT molecules were evaluated and resulted in 2-7 timeshigher potency in hBEs cell cultures compared to RTX0001.

Example 22. Stability and Efficacy Study

Buffers and cryopreservatives for freezing, concentrations, osmolalityand ionic strength were screened. Certain buffers provided stabilityacross different SORT lipids and lipid compositions. The screenedbuffers resulted in some formulations to increase in particle size aftera freeze/thaw cycle. The final particle size after a freeze/thaw cyclefor all formulation resulted in acceptable ranges for particle size (<130 nm).

Potency and tolerability of formulations were tested after being storedin frozen conditions (freeze/thaw) in in vitro and in vivo nebulizationexperiments. Lipid compositions with SORT lipids in the screened bufferunder a freeze/thaw cycle and without freeze/thaw was nebulized in hBEs.Some formulations had small change in potency (either increase ordecrease), but all formulations maintained higher potency than RTX0001.The study showed that high potency was remained in the screened bufferfor lipid compositions.

Example 23. Additional Screening Studies

Lipid compositions comprising SORT lipids were evaluated with a reduced25% and 50% total lipid / mRNA ratio (N/P ratio). The 50% mRNA reductionresulted in small decrease in potency in both hBE and mouse nebulizationfor some lipid compositions, while other tested lipid compositionsretained their potency. Two lipid compositions comprising SORT lipidswere observed to have a small increase in particle size when tested witha 50% reduction in total lipid compared to RTX0001.

The lipid compositions were screened with changes in PEG lipid contentand different N/P ratios. A small increase in particle size was observedwith decrease in PEG lipid amount. A screened range of PEG concentrationprovided 120 nm particle size.

The results provide support that a change (e.g., increase) in % PEGlipid in the lipid composition may result in a change in potency (e.g.,increase). In a hBE nebulization study, a decrease in PEG lipid amountresulted in an increased potency and an increase in PEG lipid amountresulted in a decrease in potency.

Example 24. SORT NHP Study

NHPs (Cynomolgus monkey, Macaca fascicularis, Mauritius origin, 2.5 to 3years old, male: 2.7-3.3 kg / female: 2.5-3.0 kg; N=18 total, N=8 perdose group (4 male/4 female), N=2 vehicle control (1 male/1 female) wereexamined for the efficacy of aerosol delivery by inhalation usingoronasal face mask. The delivery doses was 0.12 mg/kg or 0.24 mg/kg.Expression of DNAI1 was examined at six hours, 24 hours, 72 hours, orseven days after administration of RTX0052 (a lipid compositiondescribed herein. Readout for determining the efficacy of the wasdetermined in NHPs administered with vehicle (Group 1); low dose (Group2 with a target dose of 0.08 mg/kg; target aerosol concentration E_(c)of 0.0052 mg/L for 30 minutes); and high dose (Group 3 with a targetdose of 0.24 mg/kg; target aerosol concentration E_(c) of 0.0052 mg/Lfor 90 minutes). FIG. 18A illustrates the aerosol concentrationadministered to the NHPs, and FIG. 18B illustrates exemplarymeasurements of the doses delivered to the NHPs. FIG. 18C illustratescharacterization of the aerosol composition droplet (MMAD: mass medianaerodynamic diameter; GSDL: geometric standard deviation). The dropletcharacterization results were within recommended range of theOrganization for Economic Co-operation and Development (OECD) guidance433 for inhalation toxicity studies with an MMAD ≤ 4 µm and a GSDbetween 1.0 and 3.0.

Measurement of the droplet (lipid) in NHP blood, lung, liver, and spleentissue was determined by liquid chromatography and mass spectrometry(LC/MS-MS). Sample Matrices: Blood (plasma and blood cell fractions),Lung, Liver, Spleen. Limit of quantification (LOQ) of ionizable lipidsin plasma was 4 ng/ml. LOQ of ionizable lipid in blood cell fraction was4 ng/ml. LOQ of ionizable lipid in lung tissue cell fraction was 10ng/ml. LOQ of PEGylation of myristoyl diglyceride (DMG-PEG) in plasmawas 20 ng/ml. LOQ of DMG-PEG in blood cell fraction was 40 ng/ml. LOQ ofDMG-PEG in lung tissue was 20 ng/ml. LOQ SORT lipid in lung tissue cellfraction was 10 ng/ml. LOQ of SORT lipid in plasma was 1 ng/ml. LOQ ofPEGylation of SORT lipid in blood cell fraction was 1 ng/ml. LOQ of SORTlipid in lung tissue was 2 ng/ml. FIGS. 19A-C illustrate measurement ofLNP lipid (stemmed from aerosol droplet) in lung in both low dose andhigh dose NHP group (FIG. 19A: ionizable lipid in lung; FIG. 19B:DMG-PEG in lung; and FIG. 19C: SORT lipid). Table 12 illustratesdetection of DNAI1-HA in the processed sample of the NHP. For Table 12,2 sets of lung samples per animal (6 total) processed and assayed for:Western blot: anti-HA and anti-DNAI1; and ELISA: DNAI1-HA (Capture Ab:Anti-HA, Detection Ab: anti-DNAI1). FIG. 20A illustrates DNAI1-HAprotein expression in the NHP lung by Western blotting. FIG. 20Billustrates DNAI1-HA protein expression in the NHP lung by ELISA.

TABLE 12 DNAI1-HA detection in NHP sample Animal Group Necropsy LungSamples 3001 High Dose 0.38 mg/kg 6 h N=3 (1 ea. Caudal, Cranial, MiddleLobe) 3501 High Dose 0.38 mg/kg 6 h N=3 (1 ea. Caudal, Cranial, MiddleLobe) 3002 High Dose 0.38 mg/kg 24 h N=3 (1 ea. Caudal, Cranial, MiddleLobe) 3502 High Dose 0.38 mg/kg 24 h N=3 (1 ea. Caudal, Cranial, MiddleLobe) 3003 High Dose 0.38 mg/kg 72 h N=3 (1 ea. Caudal, Cranial, MiddleLobe) 3503 High Dose 0.38 mg/kg 72 h N=3 (1 ea. Caudal, Cranial, MiddleLobe) 1001 Vehicle 24 h N=3 (1 ea. Caudal, Cranial, Middle Lobe) 1501Vehicle 24 h N=3 (1 ea. Caudal, Cranial, Middle Lobe)

Tolerability of the RTX0052 was determined based on clinicalobservations; body and organ weights; clinical chemistry and hematology;bronchoalveolar lavage (BAL) cell differentials; cytokine and complementlevels in serum and BAL; and histopathology. There were no adverseclinical signs observed that were considered related to treatment withRTX0052. No significant changes in body weight was observed between thetreatment groups. There were also no organ weight changes (absolute andrelative to body weight) that were clearly related to RTX0052. Allchanges observed in other tissues/organs, with/without statisticalsignificance, in males and females at all dose levels were independentof dose and/or sex or were minor in magnitude or within ITR backgroundranges, thus, considered to be incidental or procedure/stress-related.FIG. 21A illustrates clinical chemistry measurements for AST, ALT, andALP. No significant changes of AST, ALT, or ALP were observed followingtreatment with RTX0052. For hematology and coagulation, there were noRTX0052-DNAI1-related changes in hematology parameters measured inmonkeys at 6-hour, 24-hour and 72-hour post end of exposure, and 7 daysof observation after inhalation exposure. Some female monkeys hadrelatively higher white blood cells and neutrophils counts in blood at 6hours, 72 hours and 7 days post end of exposure but were not consideredadverse. There were no RTX0052-DNAI1-related changes in coagulationparameters measured in monkeys at 6-hour, 24-hour and 72-hour post endof exposure, and 7 days of observation after inhalation exposure. FIG.21B illustrates the hematology counts of white blood cells andneutrophils. Some increase in neutrophils was observed in thepost-treatment measurements of both vehicle and RTX0052 groups. FIG. 21Cillustrates BAL cell differentials. For cytokine and complementanalysis, cytokines levels were measured in NHP serum and BAL. Analytesmeasured included IFN-α2a, IFN-γ, IL-1β, IL-4, IL-6, IL-10, IL-17A,IP-10, MCP-1, and TNFα. All cytokine levels were in the same range asnormal reported elves. BAL results were similarly normal as allcytokines were at or below serum lower limit of quantification (LLOQ)(except for IL-6, IL-10, and MCP-1). Table 13 illustrates the serum andBAL LLOQ measurements of analytes. FIG. 21D illustrates exemplarymeasurements of cytokine in serum. FIG. 21E illustrates exemplarymeasurements of cytokine in BAL. FIG. 21F illustrates exemplarycomplement measures of C3a and sC5b-9 measurements in plasma and serumrespectively. FIG. 21G illustrates exemplary complement measures of C3aand sC5b-9 measurements in BAL.

TABLE 13 LLOQ Analyte measurement in serum and BAL Analyte Serum LLOQ(pg/mL) BAL LLOQ (pg/mL) IFN-α2a 18.36 9.18 IFN-γ 13.38 6.69 IL-1β 2.21.1 IL-4 0.98 0.49 IL-6 1 0.5 IL-10 2.08 1.04 IL-17A 13.52 6.76 IP-104.6 2.3 MCP-1 2.66 1.29 TNFα 1.8 0.9

Based on the histopathology analysis performed herein, there was noevidence of test item-related macroscopic findings. All grossobservations were considered to be incidental, as they were sporadic andnot dose related, of low incidence, or occurred in control and treatedanimals or lacked the relevant histopathology correlates. Minimal tomild increase in the alveolar mixed cell infiltrates were observed inthe lungs of ⅜ animals treated at target total inhaled dose level of0.24 mg/kg. Due to low incidence and severity, this change wasconsidered to be potentially test item-related, but non-adverse. Allother microscopic observations were considered to be incidental,background or agonal changes, as they were of low incidence or severity,or occurred in control and test item-treated animals. Overall treatmentwas well-tolerated: No changes seen in clinical observations, clinicalchemistry, or complement measurements. Slight and transient increased inblood and BAL neutrophils was observed. All cytokine levels were withinthe normal reported range. Small transient increase in IL-6 levels wasobserved in both serum and BAL. Histopathology indicated minimal to mildincrease in the alveolar mixed cell infiltrates in ⅜ animals.

Example 25. SORT Rat Study

Rats (Sprague-Dawley (SD), 8 to 11 weeks old, male: 300-350 g / female:175-250 g; N=130 total, N=40 per dose group (20 male/20 female), N=10vehicle control (5 male/5 female)) were examined for the efficacy ofaerosol delivery by inhalation using flow-past exposure system. Thedelivery dose was low (0.25 mg/kg target dose), mid (0.49 mg/kg targetdose), or high (0.99 mg/kg target dose). Expression of DNAI1 wasexamined at six hours, 24 hours, 72 hours, or seven days afteradministration of RTX0052. Readout for determining the efficacy of theRTX0052 was determined in rats administered with vehicle (Group 1); lowdose (Group 2 with a target dose of 0.25 mg/kg; target aerosolconcentration Ec of 0.0055 mg/L for 60 minutes); mid dose (Group 3 witha target dose of 0.49 mg/kg; target aerosol concentration Ec of 0.0055mg/L for 120 minutes); and high dose (Group 4 with a target dose of 0.99mg/kg; target aerosol concentration Ec of 0.0055 mg/L for 240 minutes).FIG. 22A illustrates the aerosol concentration administered to the rats.FIG. 22B illustrates exemplary measurements aerosol homogeneity acrossthree stages. FIG. 22C illustrates the amount of doses delivered to therats. FIG. 22D illustrates characterization of the aerosol compositiondroplet (MMAD: mass median aerodynamic diameter; GSDL: geometricstandard deviation). The droplet characterization results were withinrecommended range of the Organization for Economic Co-operation andDevelopment (OECD) guidance 433 for inhalation toxicity studies with anMMAD ≤ 4 µm and a GSD between 1.0 and 3.0.

Measurement of the droplet (lipid) in rate blood, lung, liver, andspleen tissue was determined by liquid chromatography and massspectrometry (LC/MS-MS). Sample Matrices: Blood (plasma and blood cellfractions), Lung, Liver, Spleen. Limit of quantification (LOQ) ofionizable lipids in plasma was 4 ng/ml. LOQ of ionizable lipid in bloodcell fraction was 4 ng/ml. LOQ of ionizable lipid in lung tissue cellfraction was 10 ng/ml. LOQ of PEGylation of myristoyl diglyceride(DMG-PEG) in plasma was 20 ng/ml. LOQ of DMG-PEG in blood cell fractionwas 40 ng/ml. LOQ of DMG-PEG in lung tissue was 20 ng/ml. LOQ SORT lipidin lung tissue cell fraction was 10 ng/ml. LOQ of SORT lipid in plasmawas 1 ng/ml. LOQ of PEGylation of SORT lipid in blood cell fraction was1 ng/ml. LOQ of SORT lipid in lung tissue was 2 ng/ml. FIGS. 23A-Cillustrate measurement of LNP lipid (stemmed from aerosol droplet) inlung in low dose, mid dose, and high dose rat group (FIG. 23A: ionizablelipid in lung; FIG. 23B: DMG-PEG in lung; and FIG. 23C: SORT lipid).FIG. 24A illustrates DNAI1-HA protein expression in the rat lung byWestern blotting. Six out of ten lung samples I the 1.2 mg/kg, 6 hourgroup were positive for DNAI1-HA. FIG. 24B illustrates DNAI1-HA proteinexpression in the rat lung by ELISA.

Tolerability of the RTX0052 was determined based on clinicalobservations. There were no clinical signs related to treatment withRTX0052-DNAI1. No significant changes in body weight between thetreatment groups was observed. Food consumption was unaffected bytreatment with RTX0052-DNAI1. There were no organ weight changes(absolute and relative to body weight) that were clearly related toRTX0052-DNAI1. All changes observed in other tissues/organs,with/without statistical significance, in males and females at all doselevels were independent of dose and/or sex or were minor in magnitude orwithin ITR background ranges, thus, considered to be incidental orprocedure/stress-related.

FIG. 25A illustrates clinical chemistry measurements for AST, ALT, andALP in the treated rats. There were no RTX0052-DNAI1-related changes inclinical parameters measured in rats at 6-hour, 24-hour and 72-hour postend of exposure, and 7 days of observation after inhalation exposure.Some mean values differed from the control values, with and withoutstatistical significance, but the differences were independent of doseand/or sex or were minor in magnitude. Thus, they were considered tohave no biological significance. FIG. 25B illustrates the hematologycounts of white blood cells and neutrophils in the treated rats. Forhematology and coagulation, there were no RTX0052-DNAI1-related changesin hematology parameters measured in rats at 6-hour, 24-hour and 72-hourpost end of exposure, and 7 days of observation after inhalationexposure. There were no RTX0052-DNAI1-related changes in coagulationparameters measured in rats at 6-hour, 24-hour and 72-hour post end ofexposure, and 7 days of observation after inhalation exposure. Some meanvalues differed from the control values, with and without statisticalsignificance, but the differences were independent of dose and/or sex orwere minor in magnitude. Thus, they were considered to have nobiological significance. Some increase in neutrophils was observed inthe post-treatment measurements of both vehicle and RTX0052 groups. FIG.25C illustrates BAL cell differentials in the treated rats. FIG. 25Dillustrates exemplary measurements of alpha-2-macroglobulin in thetreated rats. A2M is a documented inflammation marker in the rat. Serumlevels increased 12 to 48 hours after repeated acute inflammatorystimulations. For safety evaluations, A2M wass the preferred marker forthe acute phase response in rats. No significant changes in A2M serumlevels were observed following treatment with RTX0052.

Macroscopic finding was that regardless of the time point of termination(at 6-hour, 24-hour and 72- hour post end of exposure, and 7 days postexposure) of the treated rats, all macroscopic finding in the rats wasconsidered incidental or spontaneous and not RTX0052-DNAI1-related.Microscopic finding was that regardless of the time point of termination(at 6-hour, 24-hour and 72-hour post end of exposure, and 7 days postexposure) of the treated rats, there were no microscopic pathologyfindings in the rats of this study that suggested systemic toxicity orlocal toxicity (oropharynx, nasopharynx, trachea, larynx, lungs) due toRTX0052-DNAI1. In one terminal Group 2 male rat (2016G), euthanized at 7days post end of exposure, there was multifocal panlobular hepatichemorrhagic coagulative necrosis and perilesional acute neutrophilicinflammation of the hepatic caudate lobe that correlated with itsmacroscopic finding. This microscopic finding was considered aspontaneous change that was not RTX0052-DNAI1- related, but ratherassociated with spontaneous torsion of the hepatic caudate lobe in rats(1). In another terminal Group 2 female rat (2512E), euthanized at72-hour post end of exposure, a benign subcutaneous duct cell adenomawas noted in the inguinal skin/subcutis region. This finding wasconsidered a spontaneous change that was not RTX0052-DNAI1- related,since it was not present in any of the Group 3 and Group 4 rats. Allother microscopic findings, in the treated rats of this study wereconsidered incidental or spontaneous, and not RTX0052-DNAI1-related. Allother microscopic findings in the Control rats were consideredincidental and spontaneous. LNP lipid components (ionizable lipid,DMG-PEG, and SORT lipid) were rapidly cleared from lung tissue followingtreatment. Measured levels for each in blood (plasma and cell fraction)were not detected or below the assay LOQs at each timepoint. Five out often lung samples in 1.2 mg/kg, at 6 hour post exposure were positive forDNAI1-HA protein expression by WB (FIG. 24A) and ELISA (FIG. 24B). Nosignificant changes were seen in tolerability endpoints; clinicalchemistry, hematology, BAL cell differentials, or A2M. No significanthistopathology findings that indicated local or systemic toxicity wereobserved.

Example 26. Multi-Dose Histopathology Analysis After Aerosol Delivery of3 Different LNP

Multidose administration of 4 mg of 95 % DNAI1 + 5 % Luciferase mRNAs inbuffer #27 by nebulization. Three candidate formulations (RTX0001,RTX00051, and RTX0052) were compared. Readout assays included proteindetection by ELISA, mRNA levels by qPCR/dPCR at 4our h post-last doseand post-IVIS; and lung histopathology at 72 hour and 7 days post-lastdosing. Multi-dose study evaluated toxicity of lead LNP candidates whenadministered by nebulization to mice. To deliver the LNP formulation,cages were setup for mice to acclimate for 8 days. Four groups of micewere acclimated for this study. FIG. 26A illustrates the informationrelating to the four groups of mice to be repeatedly treated withnebulization of LNP/DNAI1-HA mRNA. FIG. 26B illustrates the protocol forthe dosing, imaging, and necropsy of the repeatedly dosed mice. In vivoimaging was conducted on the treated mice. 2 mL luciferin at 30 mg/mL inPBS were nebulized with constant flow over a period of ca.4 min; 4 hpost-dosing. Animals were anesthetized with isoflurane (3% forinduction, 2% for maintenance, 1 L/min oxygen flow). Ventral images werecaptured for 1 min (binning set to 8, f stop at 1). Calibrated unitswere shown as Average Radiance (photons/s/cm2/sr) representing the fluxradiating omnidirectionally from a user defined region. Total Flux = theradiance (photons/sec) in each pixel summed or integrated over the ROIarea (cm2) x 4π. Average Radiance = the sum of the radiance from eachpixel inside the ROI/number of pixels or super pixels(photons/sec/cm2/sr).

FIGS. 27A-B illustrate whole body in vivo imaging (IVIS) of therepeatedly dosed mice. Animals, B6 Albino, male, about 7 weeks of age,naïve, were administered 4.0 mg of LNP-formulated DNAI1-HA/Luciferase bynebulization in 2 hours at 66.6 µL/min with Zero grade dry air flow at 2L/min. 4 hour post-dosing, two mice were administered 2 mL of luciferin(30 mg/mL) by nebulization and imaged on IVIS within 1-15 minpost-luciferin administration. Pseudo coloring was applied on the samescale for all images. Lung signal was plotted in graph of FIG. 27A.Whole body signal is plotted in the graph of FIG. 27B. FIG. 27Cillustrates histopathology results of the repeatedly dosed mice. Allformulations were well tolerated with most animals displaying minimal tomild inflammation scores. A single RTX0052-treated animal had moderateinflammation at 3 days post-exposure; however, this was resolved by 7days with all animals showing only minimal inflammation. Tolerabilitydata supports further studies in rats or NHPs. Histopathologic scoringsystem were as followed: 0 or normal: tissued considered to be normalunder the conditions of the study and considering the age, sex, andstrain of the animal concerned. Alterations may be present which, underother circumstances, would be considered deviation from normal; 1 orminimal: the amount of change barely exceeded that which was consideredto be within normal limits; 3 or moderate: the lesion was prominent butthere was significant potential for increased severity; limited tissueor organ dysfunction was possible; and 4 or severe: the degree waseither as complete as considered possible or great enough in intensityor extent to expect significant tissue or organ dysfunction. FIG. 27Dillustrates qPCR results showing the relative abundance of DNAI1-HAmRNA. After the last imaging of the last dose (dose 8), 2 mice per groupwere perfused. Spleen, liver and lungs were explanted. Half of eachorgan was preserved in RNAlater. Tissues were homogenized and total RNApurified with RNeasy Plus Universal Mini kit (Qiagen). Reversertranscription was performed with ProtoScript II First strand cDNAsynthesis kit (NEB). Quantitative PCR was performed and analyzed. FIG.27E illustrates Western blotting showing the protein expression ofDNAI1-HA. 25 µg protein were loaded on 4-12% Bis. Tris gel. Transferredto 0.45 µm Nitrocellulose and probed with monoclonal rat anti-HA. Theblot was stripped and reprobed with rabbit anti-DNAI1.

Example 27. Single Dose Inhalation Study

This example illustrates an exemplary experimental approaches forselecting a lipid formulation based on tolerability, biodistribution andprotein expression profiles in target cells (e.g., ciliated, club, orbasal cells) of the lungs. DNAI1 mRNA was selected with three optimized5-component formulations selected for comparison in NHPs based onstudies in Examples 24-26. A dose between 0.4 to 0.6 mg/kg was deliveredto achieve deposition in the TB region of 1-2 µg/cm² of DNAI1 mRNA for 5to 10-fold higher than estimated dose required for efficacy. Necropsytimepoints for clinical assessment were at six hours and at 72 hours.FIG. 28A illustrates delivery of 0.4 mg/kg of LNP-formulated DNAI1 mRNAby inhalation. NHPs were intubated, ventilated, and dosed for fewer than30 minutes. Targeted doses of 0/4 mg/kg were reached or exceeded for alltested LNP formulation. Presented doses of LNP-formulated DNAI1 wereestimated based on gravimetric analysis of glass fiber filters andconfirmed by filter elution and direct RNA cargo quantification usingRibogreen fluorescence assay. FIG. 28B illustrates LNP formulationaerosol characteristics. Aerosol particle size ranges for all threeformulations were appropriate for deposition in the conducting airways.FIG. 28C illustrates biodistribution of DNAI1-HA mRNA in the targetedcells. High levels of DNAI1-HA mRNA were confirmed by in situhybridization (ISH) in the lung by qPCR six hours post exposure, whileno DNAI1-HA mRNA was detected above background in spleen (at six hours),liver (at six hours), or whole blood (at 30 minutes or at 60 minutes).ISH results demonstrated that up to 30% of lung cells contained morethan 15 copies of the DNAI1-HA mRNA per cell after treatment withRTX0051 or RTX0052. FIG. 28D illustrates DNAI1-HA mRNA ISH results byH-Score. ISH results demonstrated high levels of DNAI1-HA mRNA weredelivered to lung cells with lower levels in the bronchi and trachea.FIGS. 29A-D illustrate delivery of high levels of DNAI1-HA mRNA to thelung without exposure to liver, spleen, or blood. Digital PCR was usedto measure DNAI1-HA mRNA levels in whole blood, lung, liver, and spleentissue following a single 0.4 mg/kg administration. High levels ofDNAI1-HA mRNA were detected in all three lung regions sampled at 6 hourspost-exposure with RTX0051 and RTX0052. No DNAI1-HA mRNA was detectedabove background in spleen (6 hours, FIG. 29B), liver (6 hours, FIG.29C), or whole blood (30 minutes or 60 minutes, FIG. 29D).

FIG. 30A illustrates multiplex immunofluorescent (IF) images forepithelial cell types. Epithelium cell was marked with EPCAM. Ciliatedcell was marked with acetylated-tubulin (AC-tubulin). Club cell wasmarked with secretoglobin family 1A member 1 (SCGB1A1). Goblet cell wasmarked with mucin 5AC (MUC5AC). Basal cell (stem cell) was marked withcytokeratin 5 (CK5). FIG. 30B illustrates multiplex IF analysisdemonstrating expression of DNAI1-HA protein in target cells in lung.FIG. 30C illustrates multiplex IF analysis demonstrating expression ofDNAI1-HA protein in target cells in lung with RTX0051. High sensitivityof mIF enabled detection of protein expression in tissue and cells notdetectable by other protein measurement methods. Single dose of 0.4mg/kg was administered via inhalation of LNP-formulated DNAI1-HA mRNA.Lung sections were collected from two NHPs six hours after dosing.Percentage of DNAI1-HA positive cell was calculated by combining cellcounts from all 4 examined lung sections for an individual animal. Totalnumber of cells counted per animal was about 500,000 to 1,400,000 cells.Shown are the individual data points for each treated animal and themean ± std. dev. for each group (N=2).

FIG. 30D illustrates multiplex IF analysis demonstrating expression ofDNAI1-HA protein in target cells in bronchi. FIG. 30E illustratesmultiplex IF analysis demonstrating expression of DNAI1-HA protein intarget cells in bronchi with RTX0051. Single dose of 0.4 mg/kg wasadministered via inhalation of LNP-formulated DNAI1-HA mRNA. Bronchialsections were collected from two NHPs six hours after dosing. Percentageof DNAI1-HA positive cell was calculated from a single stained sectionper animal. Total number of cells counted was about 16,000 to 65,000cells. Shown are the individual data points for each treated animal andthe mean ± std. dev. for each group (N=2). FIG. 30F illustratesmultiplex IF analysis demonstrating expression of DNAI1-HA protein intarget cells in trachea. Single dose of 0.4 mg/kg was administered viainhalation of LNP-formulated DNAI1-HA mRNA. Tracheal sections werecollected from two NHPs six hours after dosing. Percentage of DNAI1-HApositive cell was calculated from a single stained section per animal.Total number of cells counted was about 16,000 to 28,000 cells. Shownare the individual data points for each treated animal and the mean ±std. dev. for each group (N=2). FIGS. 31A-E illustrate BAL cytokine andcomplement results. FIGS. 32A-E illustrate plasma cytokine results.

FIG. 33 illustrates transient increase in neutrophils observed in BALand blood at six hours post-exposure. Transient increases in BALneutrophils and blood WBCs and neutrophils seen at six hourspost-exposure. Levels of neutrophils and WBCs had returned to baselineat 72 hours. No other significant changes in blood or BAL cellpopulations was observed. FIG. 34 illustrates selected clinicalchemistry results. Small increases were observed for AST, LDH, andcreatine kinase in individual animals after treatment. These increaseswere seen in both vehicle and TA treated animals but transient andvalues returned to baseline by 72 hours. No significant increases wereobserved for rest of the blood chemistry panel. Coagulation assaysshowed similar results for vehicle and TA treated animals. FIG. 35illustrates summary of tolerability as determined by clinicalobservations and organ weights. Animals were observed during exposure,for up to three hours post exposure and 6 hours thereafter. Cage sideclinical observation (e.g. clinical signs, etc.) was performed in the AMand PM on exposure day until euthanasia. Special attention was paid toclinical signs including but not limited to apnea, dyspnea (laboredbreathing), malaise, marked nasal discharge, lethargy, abnormalheartbeat, cyanosis, discoloration of mucous membrane, bloodystool/urine, excessive body weight loss (>20% from baseline bodyweight).No adverse reactions were reported in any of the animals during andafter exposure. No change in body weight was observed during duration ofthe study. Organ Weights and weights normalized to body weights were notstatistically different between treatment groups. Normalized lungweights appeared to be slightly higher in the animals treated withRTX0001 and RTX0052 but no statistical significance due to small samplesize. Following a single high dose administration of RTX0001, RTX0051,and RTX0052, inflammation of the lung was observed with all threeformulations. The observed severity of Grade 3 (moderate) inflammationis a concern and could impact lung function at these highconcentrations. While this degree of inflammation was seen with allformulations, the presentation was different. With RTX0001 and RTX0052,the early observation at 6 hours included Grade 3 multifocalneutrophilic alveolar inflammation, which by 72 hours had progressed tomixed cell inflammation (also Grade 3). At 72 hours, RTX0051 wasobserved with Grade 3 multifocal neutrophilic alveolar inflammation,similar to that observed at 6 hours for RTX0001 and RTX0052; it isunknown if this would have progressed to mixed cell inflammation similarto RTX0001 and RTX0052 given a later sampling.

Example 28. Single Dose Inhalation Study

This example compared 4-component (RTX0004) and 5-component (SORT,RTX0001) LNPs for lung distribution of the mRNA and protein expressionin target cells. The comparison included LNP formulation assessment; LNPformulation with mRNA as cargo (e.g., mRNA encoding DNAI1-GA), LNPformulation with modified nucleotide as cargo (e.g., mRNA comprisingmodified nucleotides); and LNP formulation with sequence optimized mRNAfor stability and translation efficiency. NHPs (Mauritius cynomolgusmacaques, 1-3 yrs. old, female, about 3 kg, N=4 per formulation, 2 pernecropsy timepoints) were intubated and ventilated and treated with theLNPs by aerosol delivery. The target delivered dose was 0.1 mg/kg.Necropsy timepoints for assessment were at six hours and at 24 hours.FIG. 36A illustrates a diagram of the aerosol delivery system. Theamount of aerosolized drug delivered past the endotracheal tube wasestimated using the test setup shown on the left. Pre-weighed glassfiber and MCE filters were attached directly at the exit of theendotracheal tube. Multiple collections were performed before, duringand after treatment of the animals. The glass filters were dried andquantified using both gravimetric analysis. The MCE filters wereanalyzed for amount of mRNA using a RiboGreen assay. FIG. 36Billustrates the results of aerosol particle size measurements. Particlesizes for test article exposure were measured for deposition in theconducting airways (branching generations 0-15 in humans).

FIG. 37 illustrates DBAI1-HA mRNA dose present in the NHP. A dashedblack horizontal line represents the targeted presented dose of 0.1mg/kg. Open yellow circles show filter collections before and afterdosing (GF, n=2) using RTX0001/DNAI1-HA mRNA. Open yellow squares showfilter collections before and after dosing (GF, n=2) usingRTX0004/DNAI1-HA mRNA. Similar results obtained for mRNA using MCEfilters and a RiboGreen assay. FIGS. 36 and 37 collectively show thataerosol particle size ranges appropriate for deposition in theconducting airways (branching generations 0-15 in humans). Targetdelivered dose of 0.1 mg/kg was achieved for RTX0001/DNAI1-HA mRNA andbetween 25-50% lower than targeted dose for RTX0004/DNAI1-HA mRNA.Problems with clogging or changes in device performance/flow rates werenot observed. Both test articles nebulized well with minimal differencesin flow rates. RTX0004/DNAI1-HA mRNA had a slightly higher flow ratecompared RTX0001/DNAI1-HA mRNA. The faster flow rates and lowerexposures for RTX0004/DNAI1-HA mRNA could be due to the larger aerosoldroplet sizes observer by APS measurements.

In situ hybridization (ISH) assay was used to detect DNAI1-HA mRNAdelivered to the lungs of NHPs using custom designed ISH probe. ISHresults were analyzed into bins: 0+: zero minimum copies/cell; 1+: oneminimum copy/cell; 2+: 4 minimum copies/cell; 3+: 10 minimumcopies/cell; and 4+: 16 minimum copies/cell. FIG. 38A illustratesDNAI1-HA mRNA ISH results for lung tissue. Data from assayqualification: 1 of 4 samples per animal analyzed. DNAI1-HA mRNAdetected in all animals. FIG. 38B illustrates that a significantfraction of lung cells contained DNAI1-HA mRNA after treatment withRTX0001 as measured by ISH and the bin scoring. FIG. 38B demonstratesthat up to 25% of lung cells contained more than 15 copies of theDNAI1-HA mRNA per cell after treatment with RTX0001. FIG. 38Cillustrates the imaging of the lung tissue used for the ISH analysis.

FIG. 39A illustrates that the delivery of high levels of DNAI1-HA to thelung did not lead to similar deliver to liver or spleen. Digital PCR wasused to measure DNAI1-HA mRNA levels in whole blood, lung, liver, andspleen tissue following a single 0.1 mg/kg administration. Primers usedwere specific for the RTX sequence optimized DNAI1-HA sequence. Highlevels of DNAI1-HA mRNA were detected in all three lung regions sampledat 6 hour post-exposure with RTX0001. In spleen and liver, DNAI1-HA mRNAwas only measured at or below the LLOQ of the assay. FIG. 39Billustrates the positive staining of DNAI1-HA tagged protein in NHPs.For RTX0001, DNAI1-HA was detected six hours or 24 hours afteradministration. Regions with higher mRNA levels correlated with regionsshowing highest levels of DNAI1-HA protein. DNAI1-HA mRNA was present inall eight treated animals. No signal detected in vehicle treatedanimals. mRNA levels were highest at six hours and lower at 24 hours.mRNA levels were highest for RTX0001 treated animals compared to RTX0004(consistent with emitted dose measurements). Using serial sections,regions with higher mRNA levels were correlated with regions showinghighest levels of DNAI1-HA protein. DNAI1-HA was detected at six hoursand 24 hours in NHPs treated with RTX0001. FIG. 39C illustratesmultiplex IF panel for key epithelial cell types. 10 NHP FFPE lungtissue blocks (1 from each animal) were used for mIF assayqualification. Two slides from each block were stained in duplicates.The cell counts of single marker positive cells, double positive cellswith DNAI1 expression, and DNAI1 MFI in double positive cells werereported. Epithelium cell was marked with EPCAM. Ciliated cell wasmarked with acetylated-tubulin (AC-tubulin). Club cell was marked withsecretoglobin family 1A member 1 (SCGB1A1). Goblet cell was marked withmucin 5AC (MUC5AC). Basal cell (stem cell) was marked with cytokeratin 5(CK5).

FIG. 40A illustrates multiplex IF panel results for NHP lung samples.DNAI1-HA was expressed in cells of the respiratory epithelium.Percentage of DNAI1-HA positive cell was calculated by combining cellcounts from 1 examined lung section per animal. DNAI1-HA expression wasdetected in lung samples from NHPs treated with RTX0001. DNAI1-HAexpression was co-localized with markers for epithelial cells, includingthe club, basal and ciliated cells (club and basal cells are precursorsfor ciliated cells). No staining detected was in lung samples from NHPstreated with RTX0004. FIG. 40B illustrates multiplex IF analysis ofexpression of DNAI1-HA protein in target cell in the lung. Single doseof 0.1 mg/kg of RTX0001/DNAI1-HA mRNA was administered via inhalation.Lung sections were collected from two NHPs at six hours and 24 hoursafter dosing. Percentage of DNAI1-HA positive cell was calculated bycombining cell counts from all 4 examined lung sections for anindividual animal. Total number of cells counted per animal was about690,000 to 1,100,000. Shown are the individual data points for eachtreated animal and the mean ± std. dev. for each group (N=2).

The present study showed that the aerosol particle size was consistentwith deposition in the conducting airways for both formulations, and theflow rates were consistent throughout exposures (more than > 0.20 mL/minfor both formulations). The exposure time for both formulations wereshort (between four to nine minutes). The biodistribution resultsindicated good distribution of mRNA, with higher levels for NHPs treatedwith formulation RTX0001. DNAI1 protein was detected in ciliated, club,and basal cells of animals treated with formulation RTX0001. Little orno DNAI1-HA expression was detected with RTX0004 (note: delivered dosewas 25-50% lower compared to RTX0001)

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is: 1-119. (canceled)
 120. A lipid nanoparticle (LNP) composition for delivering a polynucleotide to a cell in the lung wherein the LNP comprises: (i) a polynucleotide; (ii) an ionizable cationic lipid; (iii) an 1,2-Dioleoyl-3-dimethylammonium-propane (DODAP) at a molar percentage from about 20% to about 65%; (iv) a phospholipid; (v) a cholesterol; and (vi) a polyethylene glycol-conjugated lipid (PEG-lipid).
 121. The composition of claim 120, wherein the composition preferentially delivers the polynucleotide to lung cells in the subject.
 122. The composition of claim 120, wherein the ionizable cationic lipid is a compound of Formula (D-I): Core-Repeating Unit-Terminating Group (D-I), or a pharmaceutically acceptable salt thereof, wherein the core is linked to four to six repeating units and each repeating unit is linked to a nitrogen of the core, wherein: the core has the formula:

wherein: X₃ is —NR₆—, wherein R₆ is hydrogen or alkyl_((C≤8)); R₃ and R₄ are each independently amino, alkylamino_((C≤12)), dialkylamino_((C≤12)),

wherein: each e independently is 1, 2, or 3; R_(c), R_(d), and R_(f) are each independently hydrogen or alkyl_((C≤6)); c and d are each independently 1, 2, 3, 4, 5, or 6; the repeating unit is a degradable diacyl group having the formula:

wherein: A₁ and A₂ are each —O—; Y₃ is alkanediyl_((C≤12)); and R₉ is alkyl_((C≤8)), and the terminating group has the formula:

wherein: Y₄ is alkanediyl_((C≤18)); and R₁₀ is hydrogen.
 123. The composition of claim 120, wherein the ionizable cationic lipid is present in the composition at a molar percentage from about 5% to about 30%.
 124. The composition of claim 122, wherein in the compound of Formula (D-I) or the pharmaceutically acceptable salt thereof:

R₉ is —CH₃; and the terminating group is selected from the group consisting of:

.
 125. The composition of claim 124, wherein in the compound of Formula (D-I) or the pharmaceutically acceptable salt thereof, the core is linked to four repeating units and the core has the following structure:

.
 126. The composition of claim 124, wherein in the compound of Formula (D-I) or the pharmaceutically acceptable salt thereof, the core is linked to four or five repeating units and the core has the following structure:

.
 127. The composition of claim 124, wherein in the compound of Formula (D-I) or the pharmaceutically acceptable salt thereof, the core has the following structure:

.
 128. The composition of claim 120, wherein the dendrimer is

.
 129. The composition of claim 120, wherein the dendrimer is

.
 130. The composition of claim 120, wherein the dendrimer is

.
 131. The composition of claim 120, wherein the DODAP is present in the composition at a molar percentage from about 20% to about 40%.
 132. The composition of claim 120, wherein the phospholipid is present in the composition at a molar percentage from about 7.5% to about 60%.
 133. The composition of claim 120, wherein the cholesterol is present in the composition at a molar percentage from about 15% to about 46%.
 134. The composition of claim 120, wherein the PEG-lipid is present in the composition at a molar percentage from about 0.5% to about 10%.
 135. The composition of claim 120, wherein the polynucleotide comprises a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or microRNA.
 136. The composition of claim 135, wherein the polynucleotide comprises a messenger RNA (mRNA).
 137. The composition of claim 136, wherein the mRNA encodes a gene-editing system or component thereof.
 138. The composition of claim 136, wherein the polynucleotide comprises modified mRNA by 5-uracil (pseudouracil) and/or capping reaction.
 139. The composition of claim 136, wherein the mRNA comprises a sequence encoding cystic fibrosis transmembrane conductance regulator (CFTR).
 140. The composition of claim 136, wherein the mRNA comprises a sequence encoding dynein axonemal intermediate chain 1 (DNAI).
 141. The composition of claim 120, wherein the composition is an aerosolized composition.
 142. A method for treating a lung disease in a subject in need thereof, comprises administering a lipid nanoparticle (LNP), wherein the LNP comprises: (i) a polynucleotide (ii) an ionizable cationic lipid; (iii) an DODAP at a molar percentage from about 20% to about 65%; (iv) a phospholipid; (v) a cholesterol; and (vi) a polyethylene glycol-conjugated lipid (PEG-lipid), wherein the ionizable cationic lipid is a compound of Formula (D-I): Core-Repeating Unit-Terminating Group (D-I), or a pharmaceutically acceptable salt thereof, wherein the core is linked to four to six repeating units and each repeating unit is linked to a nitrogen of the core, wherein: the core has the formula:

wherein: X₃ is —NR₆—, wherein R₆ is hydrogen or alkyl_((C≤8)); R₃ and R₄ are each independently amino, alkylamino_((C≤12)), dialkylamino_((C≤12)),

wherein: each e independently is 1, 2, or 3; R_(c), R_(d), and R_(f) are each independently hydrogen or alkyl_((C≤6)); c and d are each independently 1, 2, 3, 4, 5, or 6; the repeating unit is a degradable diacyl group having the formula:

wherein: A₁ and A₂ are each -O-; Y₃ is alkanediyl_((C≤12)); and R₉ is alkyl_((C≤8)), and the terminating group has the formula:

wherein: Y₄ is alkanediyl_((C≤18)); and R₁₀ is hydrogen.
 143. The method of claim 142, wherein the administering comprises administration by inhalation.
 144. The method of claim 142, wherein the administering comprises administration by intravenous injection.
 145. The composition of claim 120, wherein the DODAP is present in the composition at a molar percentage from about 20% to about 40%; wherein the phospholipid is present in the composition at a molar percentage from about 7.5% to about 60%; wherein the cholesterol is present in the composition at a molar percentage from about 15% to about 46%; wherein the PEG-lipid is present in the composition at a molar percentage from about 0.5% to about 10%; and wherein the dendrimer is 4A3-SC7, 4A3-SC8, or 5A2-SC8. 